Comparative electron microscopy of chorio-allantoic placental barrier in some Indian Chiroptera

In the present study the comparative ultrastructure of the definitive chorio-allantoic placental barrier has been studied in considerable detail in six species of bats, representing six different families and both suborders of Chiroptera, by electron microscopy, and these species illustrate different kinds of interhaemal membranes met with among bats. The definitive chorio-allantoic placenta of Rousettus leschenaulti is haemodichorial, since the syncytiotrophoblast and cytotrophoblast layers are present to term. The fine structure of the placental barrier in the labyrinth of the definitive placenta of Rhinopoma hardwickei hardwickei is essentially endotheliomonochorial due to the presence of a single layer of cytotrophoblast and maternal endothelial cells. The placenta of Taphozous melanopogon, examined electron-microscopically in the present study, shows a thick maternal endothelium, a continuous interstitial membrane and the presence of a single layer of syncytiotrophoblast. The placenta of Megaderma comprises a typical endotheliochorial labyrinth and the presence of two layers of trophoblast. In Rhinolophus rouxi, the mature placenta during advanced pregnancy resembles that of Megaderma, its labyrinth containing large maternal capillaries with maternal endothelial cells and the two layers of trophoblast. Finally, the placental barrier of Hipposideros fulvus fulvus is haemodichorial due to the presence of two layers of trophoblast and the absence of maternal endothelial cells.     

Proliferation of villous trophoblast of the human placenta in normal and abnormal pregnancies.

The proliferation of villous trophoblast in the human placenta was estimated throughout normal gestation and in term placentae from preeclamptic and smoking mothers by two different methods. These were: 1) labeling of DNA producing cells by bromodeoxyuridine (BrdU) followed by immunohistochemistry using a monoclonal anti-BrdU antibody, and 2) immunohistochemical identification of all proliferating cells by the monoclonal antibody Ki67. Both methods revealed comparable results. In uncomplicated pregnancies there was a remarkable decrease in the labeling indices from early gestation to term. This was the result of a diminution of the number of Langhans' cells, although the cell division rate within the Langhans' cell layer remained nearly constant throughout gestation. A prolongation of the cell cycle in the cytotrophoblast cells at term was indicated by an increase in the Ki67/BrdU ratio. Compared with normal term placentae, there was an increase in the trophoblast proliferation rate in preeclampsia, but not in placentae from smoking mothers. Moreover, the number of Langhans' cells was diminished in placentae from smokers. The results indicate that there are different pathogenetic mechanisms of placental impairment in preeclampsia and in maternal smoking. In preeclampsia an injury to the syncytiotrophoblast seems to lead to a repair hyperplasia of the cytotrophoblast, whereas in maternal smoking, there seems to be a direct toxic effect on the cytotrophoblastic cells.     

Ultrastructural development of chorioallantoic placenta in the Indian Miniopterus bat, Miniopterus schreibersii fuliginosus (Hodgson).

The chorioallantoic placental interhemal membrane of Miniopterus schreibersii fuliginosus has been described electron-microscopically. Morphologically there are three main types of placentae which develop in chronological sequence. They are (1) primary placenta, (2) secondary placenta and (3) tertiary placenta. In neural groove and limb-bud embryos the primary placenta consists of the following elements which separate the maternal and fetal circulations: (1) a continuous ectoplasmic layer, (2) intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The primary placenta degenerates until term when it consists of a thin syncytiotrophoblastic layer resting on basal lamina. Mesenchyme does not show the presence of fetal capillaries. The secondary placenta is formed in early limb-bud embryos. The electron microscope has revealed that the placenta is of the endotheliomonochorial type and (1) consists of a well-developed maternal endothelium, (2) the trophoblast surrounding the maternal blood tubule is cellular, not syncytial as previously thought and the apical plasma membrane of these trophoblastic cells is in direct contact with the discontinuous interstitial membrane, (3) basal lamina, (4) mesenchyme and (5) fetal endothelium. Tertiary placenta at full term stage is of the hemodichorial type having the following elements: (1) thin ectoplasmic layer, (2) a thick intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The definitive chorioallantoic placental barrier in this bat thus differs from the organization earlier proposed by Chari and Gopalakrishna [Proc. Indian Acad. Sci. 93: 463-483, 1984] on the basis of light-microscopic observations: (1) the absence of maternal endothelium in the primary placenta from the neural groove and early limb-bud embryos, (2) the existence of only cellular trophoblast in the secondary placenta throughout the gestation and (3) the presence of well-developed hemodichorial tertiary placenta is the unique feature of the interhemal membrane in higher Chiroptera.     

Differential expression of the receptors for epidermal growth factor and insulin in the developing human placenta

The detailed cellular distribution of epidermal growth factor (EGF) receptors and insulin receptors during the development of the human placenta was examined. We show that EGF receptors are expressed by villous cytotrophoblast cells in first trimester human placentae. However, where these cells proliferate to form extravillous cytotrophoblast cell columns, there is a dramatic decrease in EGF receptor expression. There is no such differential expression of insulin receptors on this cell population. In contrast, both EGF-and insulin-receptors are present throughout gestation on the microvillous membrane of the terminally differentiated and non-proliferative syncytiotrophoblast although, at term, EGF-but not insulin-receptors are also found on the basolateral membrane of this epithelium. We further show that EGF receptors isolated from first trimester and term human placentae have functional tyrosine kinase activities but differ in their extent of glycosylation. These results suggest that EGF receptors probably play several distinct functional roles in these epithelial cells depending on their proliferative capacity and differentiation status.     

Early placentation in the African green monkey (Cercopithecus aethiops)

Placentation in the green monkey (Cercopithecus aethiops) from gestational ages of 18-30 days was studied by light and electron microscopy. The placentae show many features in common with that of the rhesus monkey and other related macaques. Implantation is superficial and bidiscoid, and the placenta is classified as villous and hemomonochorial. Epithelial plaque formation and a moderate stromal reaction occur. Plaque cells are large, with massive deposits of glycogen and marginal pleomorphic dense bodies. Observations further support the concept that chorionic blood vessels, angioblastic and mesenchymal cells are not derived by delamination from cytotrophoblast. Capillary formation is by attachment of one or more mesenchymal cells and/or their processes. Embryonic blood was not observed in the villi until after day 22.     

X-chromosome inactivation in the human cytotrophoblast

Preferential paternal X-chromosome inactivation occurs in the cell lineages that differentiate first within the female rodent blastula (trophectoderm and extraembryonic endoderm). The present studies were designed to test the nature of X-chromosome inactivation (XCI) in the earliest differentiating cell lineage of the human placenta, the cytotrophoblast. Using glucose-6-phosphate-dehydrogenase (G6PD) polymorphisms as a marker system, term placentae were obtained from 13 female heterozygotes where parental allelic contributions could be determined. Chorionic villi were enzymatically digested and centrifuged in a Percoll density gradient to isolate a pure population of cytotrophoblasts, which was ascertained by cell culture, differentiation to syncytiotrophoblasts, and histochemical staining for alpha-human chorionic gonadotrophin (alpha-HCG). On electrophoresis, all 13 samples exhibited exclusive or near exclusive expression of the maternally derived X-linked enzyme variant, regardless of whether it was G6PD A or G6PD B. No intermediate bands were seen, indicating a single active G6PD locus per cell. The stromal cells of the villi, which derive from the mesoderm and differentiate later than the cytotrophoblast, exhibit random XCI. These findings establish preferential paternal XCI in the cytotrophoblast, the cell type first to differentiate within the human blastula.     

Hemochorial placentation in the primate: expression of vascular endothelial growth factor, angiopoietins, and their receptors throughout pregnancy

Vascular development and its transformation are necessary for successful hemochorial placentation, and vascular endothelial growth factor (VEGF), angiopoietins, and their receptors may be involved in the molecular regulation of this process. To determine the potential role of these putative regulators in a widely studied primate, the common marmoset, we investigated their mRNA expression and protein location in the placenta throughout pregnancy using in situ hybridization, Northern blot analysis, and immunocytochemistry. VEGF was localized in decidual and cytotrophoblast cells, and its highest expression was found in the maternal decidua. The Flt receptor was exclusively detected in the syncytial trophoblast with increasing expression in placentae from 10 wk to term. Soluble Flt (sFlt) was also detectable by Northern blot analysis. KDR receptor expression was restricted to mesenchymal cells during early placentation and to the fetoplacental vasculature during later placentation. KDR expression increased throughout pregnancy. Angiopoietin-1 (Ang-1) was localized in the syncytial trophoblast, being highly expressed in the second half of gestation. Ang-2 mRNA localized exclusively to maternal endothelial cells, and was highly expressed in 10-wk placentae. The Tie-2 receptor was found in cytotrophoblast cells and in fetal and maternal vessels. High Tie-2 levels were detected in the wall of chorion vessels at 14-wk, 17-wk, and term placentae. These results suggest that the processes of trophoblast invasion, maternal vascular transformation, and fetoplacental vascular differentiation and development are regulated by the specific actions of angiogenic ligand-receptor pairs. Specifically, 1) VEGF/Flt and Ang-1/Tie-2 may promote trophoblast growth, 2) VEGF/KDR and Ang-1/Tie-2 may support fetoplacental vascular development and stabilization, 3) sFlt may balance VEGF actions, and 4) Ang-2/Tie-2 may remodel the maternal vasculature.     

Aggregation of dispersed human cytotrophoblastic cells: lessons relevant to the morphogenesis of the placenta

The syncytial trophoblast of the human placenta forms by the fusion of mononuclear cytotrophoblast cells. Cytotrophoblast cells only fuse with other trophoblastic cells, indicating a specificity to this interaction. To explore the cellular aggregation which precedes fusion, we examined the association of cytotrophoblast cells isolated from term placentae and JEG-3 choriocarcinoma cells, a cytotrophoblast-like cell line, in suspension culture. Cytotrophoblast cells were isolated by dispersion of chorionic villi in trypsin-DNase in Ca2+/Mg2(+)-free medium. JEG-3 cells were released from culture flasks by trypsinization in Versene-EDTA buffer. In suspension culture, each cell type aggregated forming tissue-like masses over a 24-hr period. Transmission electron microscope analysis demonstrated the formation of numerous desmosomes between the aggregated cells. In outgrowth culture, the aggregates created in suspension were maintained as microvilli-covered multicellular structures with hollow cores. The extent of aggregation was dependent upon the concentration of cells in the incubations with greater aggregation occurring with higher cell densities. Aggregation of both cytotrophoblast cells and JEG-3 cells progressed rapidly during the initial 10 hr of incubation and then continued at a slower rate. Aggregation took place in serum-containing and serum-free medium, but was impeded in Ca2+/Mg2(+)-free medium. Incubation of JEG-3 and cytotrophoblast cells in the presence of the protein synthesis inhibitor, cycloheximide, prevented aggregation, whereas the inhibitor of N-linked glycosylation, tunicamycin, did not. The inhibitor of RNA synthesis, actinomycin D, had no effect on the aggregation of the cells during the initial 6 hr of aggregation. These findings suggest that trypsin treatment in Ca2+/Mg2(+)-poor medium removed a protein(s) from the trophoblast cell surface which must be resynthesized for cell-cell association to take place.     

Giant cells in the placenta of the bactrian camel in the fetal period of development]

Polyploid giant cells resulting from differentiation of normal cytotrophoblast cells were found in the composition of the placenta cytotrophoblast of the Bactrian camel. In the placenta of the Bactrian camel the transformation of cytotrophoblast cells into multinuclear giant cells is realized via endomitotic polyploidization and distinctly reflects active synthetic processes occurring in these cells related with the trophic function of the placenta. It may be supposed that giant cells of the placenta of female Bactrian camels also participate in the endocrinous function of the placenta. Their histolitical function is completely excluded.     

Expression and localization of estrogen receptor-alpha protein in normal and abnormal term placentae and stimulation of trophoblast differentiation by estradiol

Estrogens play an important role in the regulation of placental function, and 17-beta-estradiol (E2) production rises eighty fold during human pregnancy. Although term placenta has been found to specifically bind estrogens, cellular localization of estrogen receptor alpha (ER-alpha) in trophoblast remains unclear. We used western blot analysis and immunohistochemistry with h-151 and ID5 monoclonal antibodies to determine the expression and cellular localization of ER-alpha protein in human placentae and cultured trophoblast cells. Western blot analysis revealed a ~65 kDa ER-alpha band in MCF-7 breast carcinoma cells (positive control). A similar band was detected in five normal term placentae exhibiting strong expression of Thy-1 differentiation protein in the villous core. However, five other term placentae, which exhibited low or no Thy-1 expression (abnormal placentae), exhibited virtually no ER-alpha expression. In normal placentae, nuclear ER-alpha expression was confined to villous cytotrophoblast cells (CT), but syncytiotrophoblast (ST) and extravillous trophoblast cells were unstained. In abnormal placentae no CT expressing ER-alpha were detected. Normal and abnormal placentae also showed ER-alpha expression in villous vascular pericytes and amniotic (but not villous) fibroblasts; no staining was detected in amniotic epithelial cells or decidual cells. All cultured trophoblast cells derived from the same normal and abnormal placentae showed distinct ER-alpha expression in western blots, and the ER-alpha expression was confined to the differentiating CT, but not to the mature ST. Trophoblast cells from six additional placentae were cultured in normal medium with phenol red (a weak estrogen) as above (PhR+), or plated in phenol red-free medium (PhR-) without or with mid-pregnancy levels of E2 (20 nM). Culture in PhR- medium without E2 caused retardation of syncytium formation and PhR-medium with E2 caused acceleration of syncytium formation compared to cultures in PhR+ medium. These data indicate that the considerable increase in estrogen production during pregnancy may play a role, via the ER-alpha, in the stimulation of CT differentiation and promote function in normal placentae. This mechanism, however, may not operate in abnormal placentae, which show a lack of ER-alpha expression.     

Uterine epithelial changes during placentation in the viviparous skink Eulamprus tympanum

We used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to describe the complete ontogeny of simple placentation and the development of both the yolk sac placentae and chorioallantoic placentae from nonreproductive through postparturition phases in the maternal uterine epithelium of the Australian skink, Eulamprus tympanum. We chose E. tympanum, a species with a simple, noninvasive placenta, and which we know, has little net nutrient uptake during gestation to develop hypotheses about placental function and to identify any difference between the oviparous and viviparous conditions. Placental differentiation into the chorioallantoic placenta and yolk sac placenta occurs from embryonic Stage 29; both placentae are simple structures without specialized features for materno/fetal connection. The uterine epithelial cells are not squamous as previously described by Claire Weekes, but are columnar, becoming increasingly attenuated because of the pressure of the impinging underlying capillaries as gestation progresses. When the females are nonreproductive, the luminal uterine surface is flat and the microvillous cells that contain electron-dense vesicles partly obscure the ciliated cells. As vitellogenesis progresses, the microvillous cells are less hypertrophied than in nonreproductive females. After ovulation and fertilization, there is no regional differentiation of the uterine epithelium around the circumference of the egg. The first differentiation, associated with the chorioallantoic placentae and yolk sac placentae, occurs at embryonic Stage 29 and continues through to Stage 39. As gestation proceeds, the uterine chorioallantoic placenta forms ridges, the microvillous cells become less hypertrophied, ciliated cells are less abundant, the underlying blood vessels increase in size, and the gland openings at the uterine surface are more apparent. In contrast, the yolk sac placenta has no particular folding with cells having a random orientation and where the microvillous cells remain hypertrophied throughout gestation. However, the ciliated cells become less abundant as gestation proceeds, as also seen in the chorioallantoic placenta. Secretory vesicles are visible in the uterine lumen. All placental differentiation and cell detail is lost at Stage 40, and the uterine structure has returned to the nonreproductive condition within 2 weeks. Circulating progesterone concentrations begin to rise during late vitellogenesis, peak at embryonic Stages 28-30, and decline after Stage 35 in the later stages of gestation. The coincidence between the time of oviposition and placental differentiation demonstrates a similarity during gestation in the uterus between oviparous and simple placental viviparous squamates.     

Growth factor-extracellular matrix synergy in the control of trophoblast invasion

At the periphery of the human placenta, trophoblast attaches to the uterine wall. The tissue interface contains many anchoring sites, with cytotrophoblast columns that form bridges between the overlying extraembryonic (villous) mesenchyme and the maternal decidual stroma beneath. From the periphery of these columns, large numbers of trophoblast cells detach, migrate through the decidua and eventually colonize and transform maternal arteries. In this way the placenta increases and gives priority to the maternal blood supply to the conceptus. We have shown that when early villous tissue is explanted on a collagen gel in serum-free medium, anchoring-site morphogenesis occurs. Thus, in the presence of placental mesenchyme but in the absence of maternal cells, contact with a permissive extracellular matrix (ECM) is necessary and sufficient for cytotrophoblast column development. Proliferation of trophoblast occurs, followed by differentiation into a columnar cell phenotype in which cells remain attached to one another and to the ECM. At this stage, interaction between fibronectin and integrin alpha5beta1 at the cell surface stabilizes the column and the cells remain as a contiguous multilayered sheet. However, the addition of serum-free conditioned medium from first-trimester placental fibroblasts stimulates cytotrophoblast to detach from the distal column and migrate in streams across the ECM. The removal of insulin-like growth factor I (IGF-I) from the fibroblast medium decreases streaming activity, whereas the addition of exogenous IGF-I (10 ng/ml) to serum-free medium produces a streaming phenotype. In contrast, transforming growth factor beta1 (10 ng/ml) maintains the cells in a tight sheet. These results suggest the possibility of a paracrine interaction between villous mesenchyme and cytotrophoblast in anchoring sites to stimulate the infiltration of the maternal ECM by trophoblast. Such a mechanism would be self-limiting because the signal diminishes with distance from the placenta.     

Distribution of mosaicism in human placentae

Traditional first trimester chorionic villus sampling (CVS) for prenatal diagnosis can be performed by cytogenetic analysis of cytotrophoblast or chorionic villous stroma. Approximately 2% of pregnancies studied by CVS show confined placental mosaicism (CPM) involving either cytotrophoblast, stroma or both. We present the results of a cytogenetic study of nine term placentae from pregnancies with prenatally diagnosed CPM. The aneuploid cell lines involved trisomies for chromosomes 7,9,16, and X. The cytotrophoblast and villous stroma from multiple biopsies of these placentae were examined using a combination of interphase and metaphase cytogenetic analysis. CPM was detected in all nine of the term placentae and both tissue-specific and site-specific patterns of mosaicism could be discerned. These results indicate that the analysis of villous stroma and cytotrophoblast from multiple placental biopsies is necessary to improve our understanding of the evolution of CPM during pregnancy and its effect on the fetus. Received: 1 May 1995 / Revised: 11 August 1995     

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) mRNA expression and localization and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) in human placenta of the first, second, and third trimester

The cellular localization of protein tyrosine phosphatase 51 (PTPIP51) and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) was studied in human placentae of different gestational stages. The expression of PTPIP51 protein and mRNA was observed in the syncytiotrophoblast and cytotrophoblast layer of placentae from the first, second, and third trimesters. In contrast, PTP1B expression was restricted to the syncytiotrophoblast during all gestational stages. Cells of the cytotrophoblasts and parts of the syncytiotrophoblasts expressing high amounts of PTPIP51 were found to execute apoptosis as shown by TdT-mediated dUTP-biotin nick end labeling assay, cytokeratin 18f, and caspase 3 expression. PTPIP51 could also be traced in the endothelium and smooth muscle cells of placental arterial and venous vessels, identified by double immunostainings with antibodies directed against van Willebrand factor and alpha-smooth muscle actin. Some of these cells showing a high PTPIP51 reactivity were Ki67 positive, indicating proliferation. Additionally, a small population of placental CD14-positive macrophages and mesenchymal cells within the villous stroma were detected as PTPIP51 positive. Our data suggest that both proteins, PTPIP51 and PTP1B, play a role in differentiation and apoptosis of the cytotrophoblast and syncytiotrophoblast, respectively. Moreover, PTPIP51 may also serve as a cellular signaling partner in angiogenesis and vascular remodeling.     

Real-Time Tracking of BODIPY-C12 Long-Chain Fatty Acid in Human Term Placenta Reveals Unique Lipid Dynamics in Cytotrophoblast Cells

While the human placenta must provide selected long-chain fatty acids to support the developing fetal brain, little is known about the mechanisms underlying the transport process. We tracked the movement of the fluorescently labeled long-chain fatty acid analogue, BODIPY-C₁₂, across the cell layers of living explants of human term placenta. Although all layers took up the fatty acid, rapid esterification of long-chain fatty acids and incorporation into lipid droplets was exclusive to the inner layer cytotrophoblast cells rather than the expected outer syncytiotrophoblast layer. Cytotrophoblast is a progenitor cell layer previously relegated to a repair role. As isolated cytotrophoblasts differentiated into syncytialized cells in culture, they weakened their lipid processing capacity. Syncytializing cells suppress previously active genes that regulate fatty-acid uptake (SLC27A2/FATP2, FABP4, ACSL5) and lipid metabolism (GPAT3, LPCAT3). We speculate that cytotrophoblast performs a previously unrecognized role in regulating placental fatty acid uptake and metabolism.     

Extracellular matrix composition and hypoxia regulate the expression of HLA-G and integrins in a human trophoblast cell line

During human placentation, extravillous cytotrophoblast cells emerge from chorionic villi contacting the decidua to invade the uterine wall. When isolated from first-trimester placentae, cytotrophoblast cells undergo step-wise differentiation in vitro that recapitulates the phenotypic heterogeneity observed in vivo. We examined a cell line, HTR-8/SVneo, that has been established from human first-trimester cytotrophoblast to determine whether these cells possess some of the unique cytotrophoblast characteristics that have been described previously. Exposure during serum-free culture to hypoxic conditions (2% oxygen concentration) increased HTR-8/SVneo cell proliferation and reduced invasion of a three-dimensional basement membrane (Matrigel). During culture on surfaces coated with individual extracellular matrix proteins, HTR-8/SVneo cells expressed cytokeratin but not the trophoblast-specific major histocompatibility protein, HLA-G. However, HLA-G expression was induced in HTR-8/SVneo cells that contacted Matrigel. Expression of the alpha5 integrin subunit was relatively unaffected by matrix composition, whereas alpha1 was up-regulated and alpha6 was down-regulated after transferring cells to Matrigel. Hypoxia increased alpha6 and decreased both alpha1 and HLA-G expression on Matrigel. HTR-8/SVneo cells retain several important characteristics associated with primary cultures of first-trimester human cytotrophoblast cells, including their altered behavior in response to a changing maternal environment.     

Ultrastructure of the full-term shark yolk sac placenta. I. Morphology and cellular transport at the fetal attachment site

During ontogeny, the yolk sac of some viviparous sharks differentiates into a yolk sac placenta that persists to term. The placenta is non-invasive and non-deciduate. Hematrophic transport is the major route of nutrient transfer from mother to fetus. The placental unit consists of: (1) an umbilical stalk; (2) the smooth, proximal portion of the placenta; (3) the distal, rugose portion; (4) the egg envelope; and (5) the maternal uterine tissues. Exchange of metabolites is effected through the intervening egg envelope. The distal rugose portion of the placenta is the fetal attachment site. It consists of: (1) surface epithelial cells; (2) a collagenous stroma with vitelline capillaries; and (3) an innermost boundary cell layer. The columnar surface epithelial cells are closely apposed to the inner surface of the egg envelope. Wide spaces occur between the lateral margins of adjacent cells. Surface epithelial cells contain an extensive apical canalicular-tubular system and many whorl-like inclusions in their basal cytoplasm. Capillaries of the vitelline circulation are closely situated to these cells. A well-developed collagenous stroma separates the surface epithelium from an innermost boundary cell layer. In vitro exposure of full-term placentae to solutions of trypan blue and horseradish peroxidase (HRP) reveals little uptake by the smooth portion of the placenta but rapid absorption by the surface epithelial cells of the distal, rugose portion. HRP enters these cells by an extensive apical system of smooth-walled membranous anastomosing canaliculi and tubules. Prominent whorl-like inclusions that occupy the basal cytoplasm of the surface cells, adjacent to the pinocytotically active endothelium of the vitelline capillaries, are hypothesized to be yolk proteins that are transferred from the mother to embryo throughout gestation.