Placentation in the degu (Octodon degus): analogies with extrasubplacental trophoblast and human extravillous trophoblast

This study examined the placentation in the degu, the origin of the extrasubplacental trophoblast (EST) (extravillous trophoblast in human), and the activity of Na+/K+ ATPase in the placental barrier during different gestational ages, as part of a wider effort to understand the reproductive biology of this species. Fifteen degus at the first stage of gestation, midgestation and at term of pregnancy were studied. At day 27 of gestation, the subplacenta is formed under the wall of the central excavation. Simultaneously, the outermost trophoblast of the ectoplacental cone differentiated into secondary trophoblast giant cells that lie on the outside of the placenta, forming an interface with the maternal cells in the decidua. These giant cells immunostained positive for cytokeratin (CK) and placental lactogen (hPL) until term. During this period, the EST merged from the subplacenta to the decidua and immunostained negative for CK, but at term, immunostained for CK and hPL in the maternal vessels. The vascular mesenchyme of the central excavation invaded the chorioallantoic placenta during this period, forming two fetal lobules of labyrinthine-fine syncytium, the zone of the placental barrier. The activity of Na+/K+ ATPase in the placental barrier was constant during the gestational period. The residual syncytium at the periphery of the placental disc and between the lobules was not invaded by fetal mesenchyme and formed the marginal and interlobular labyrinthine syncytium that immunostained first for CK, and later for hPL, as in the labyrinthine fine syncytium. The presence of intracytoplasmic electron-dense material in the interlobular labyrinthine syncytium suggested a secretory process in these cells that are bathed in maternal blood. Placentas obtained from vaginal births presented a large, single lobe, absence of the subplacenta, and a reduced interlobular labyrinthine syncytium. At day 27, the inverted visceral yolk sac is observed and its columnar epithelium immunostained for CK and hPL. This suggests that the yolk sac is an early secretory organ. The epithelium of the parietal yolk sac covers the placenta. The origin of the EST in the degu placenta and its migration to maternal vessels allows us to present this animal model for the study of pregnancy pathologies related to alterations in the migration of the extravillous trophoblast.     

The fine structure of the chinchilla placenta.

The structure of the placental labyrinth, interlobular or "coarse" syncytium, visceral (splanchnopleuric) yolk sac, giant cells and subplacenta of the chinchilla was studied with the electron microscope. The fine structure of the interhemal membrane of the placental labyrinth was found to be hemomonochorial, consisting of a single layer of syncytial trophoblast. In this respect, the placental labyrinth was similar to that of another caviomorph rodent, the guinea pig. The labyrinthine trophoblast had pinocytotic vesicles as well as larger vaculoes and multivesicular bodies. The interlobular syncytium contained granular endoplasmic reticulum, and in one case from early in gestation there were intracisternal granules in the ER. The visceral endodermal cells of the inverted yolk sac placenta had a well-developed system of apical vesicles and tubules as well as larger cytoplasmic vacuoles. Their appearance was similar to that of endodermal cells found in other rodents which are known to absorb proteins and other substances from the uterine lumen. Towards term the giant cells were often vacuolated and contained large deposits of glycogen as well as lipid droplets. The syncytial trophoblast of the subplacenta contained numerous moderately electron-dense granules which may be secretory in function; cytotrophoblastic cells lacked these granules. The subplacental syncytium often surrounded spaces or lacunae which contained an electron-dense granular material.     

Development of trophoblast and placenta of the mouse. A reinvestigation with regard to the in vitro culture of mouse trophoblast and placenta

At 5 days post conceptionem (p.c.) shortly after implantation, giant cell transformation starts at the abembryonic pole of the blastocyst, spreading over the mural trophoblast; 1 day later, the first ectoplacental giant cells appear at the base of the fast growing ectoplacental cone (derived from the polar trophoblast). Giant cell transformation expands over it periphery. Thus, by the 8th day p.c., the conceptus is separated from the maternal tissue by a continuous layer of giant cells, variable in thickness. Giant cells reach their greatest size by 10 days p.c. in the mural tophoblast and by 12 days p.c. in the chorioallantoic placenta. They are probably no longer formed after that stage. Around the 8th day p.c., the allantois reaches contact with the ectoplacental cone, which develops into the chorioallantoic (definitive) placenta. At 9 days p.c., its four zones can already be discriminated: chorionic plate, labyrinth, junctional zone (trophospongium), and zone of giant cells, respectively. Within the next day, the chorioallantoic placental circulation is established. The yolk sac placental circulation is established by the 9th day p.c. The villi of the proximal layer of the yolk sac increase in size and number, and their capillary network becomes more dense until the 12th to 14th day p.c. This provides evidence that the yolk sac placenta exerts its function--to a certain extent--beyond the establishment of the definitive placenta. Around the 14th day p.c., the placental labyrinth reaches its definitive features. Fetal capillaries in the labyrinth, branching from unbilical blood vessels within the septa of connective tissue are surrounded by trophoblast cells. They form a dense vascular network bathing in maternal blood. The structures of the placental zones remain almost the same during further development, the borders becoming sometimes little blurred. Adjacent to the chorionic plate, subchorionic clefts appear at the 14th day p.c. These clefts become confluent to form the intraplacental space, regularly communicating with the yolk sac cavity. At the end of gestation (19th day p.c.) there is a considerable amount of eosinophilic material ('fibrinoid') between the zone of giant cells and the decidua, probably produced by the giant cells.     

The subplacenta of the red-rumped agouti (Dasyprocta leporina L)

Background  

Hystricognath rodents have a lobed placenta, comprising labyrinthine exchange areas and interlobular trophoblast. These correspond to the labyrinthine and spongy zones of other rodent placentae. Beneath them, however, is a structure unique to hystricognath rodents called the subplacenta. We here describe the subplacenta of the red-rumped agouti and examine the possible functional correlates of this structure.     

Immunohistochemical identification of the extravillous trophoblast during the placentation of the degu (Octodon degus)

Recent data indicate that placentation in Octodon degus is similar to that in humans, making it a potential animal model for studies in human placental pathologies related to alterations in the migration of the extravillous trophoblast (EVT). Our objective was to immunohistochemically identify degu EVT during placentation by using cytoskeletal protein markers to establish the normal migratory pattern of the EVT. Fifteen O.degus were divided into three equal groups: day 27, 60, and 84 of gestation. The placentas were immunostained for cytokeratin (CK) and alpha smooth muscle actin (SMA). At day 27, the migrating EVT immunostained for SMA but not for CK. Once the EVT was incorporated in the maternal vessels (day 60) it was positive for CK but negative for SMA. The smooth muscle cells of the mesometrial arteries that remained after EVT invasion were positive for SMA. At day 84, the media muscular layer had partially regenerated but some EVT was still present. Furthermore, at day 27 cyclooxygenase-1 (COX-1) was detected in the endothelium of the maternal decidual vessels. Our results suggest that during the early stages of placentation, the cytoskeletal organization of the actin network of the migrating EVT corresponds to that of a cell with motile behavior. Once the EVT invaded the spiral arteries, the cytoskeleton reorganized, adopting the structure of an epithelial-like cell, expressing CK intermediate filaments. The media muscle layer regenerated near the end of gestation but some EVT remained. During EVT formation the endothelium of the maternal decidual vessels immunostained for COX-1.     

Morphogenesis of the foetal membranes and placenta of the root-rat (Tachyoryctes splendens (Rüppel))

The morphogenesis of the foetal membranes and placenta of the root-rat Tachyoryctes splendens was studied by light microscopy. Implantation of the blastocyst is antimesometrial. The mode of implantation is secondary interstitial and the decidual reaction is similar to that in other rodents. Amniogenesis is by cavitation with a temporary open epamniotic cavity. Inversion of the yolk sac is complete but the disappearance of the parietal segment occurs relatively late. With the breakdown of both the decidua capsularis and parietalis, the yolk sac wall near the margins of the placenta becomes bathed in a pool of maternal blood and tissue debris resembling the haemophagous organ. The yolk sac villi are well vascularized. The allantoic cavity is persistent up to the limb bud stage. The definitive placenta is haemochorial and shows conspicuous endodermal sinuses or placental pits.     

Function and control of human invasive trophoblast subtypes: Intrinsic vs. maternal control

ABSTRACT

The establishment of a functional placenta is pivotal for normal fetal development and the maintenance of pregnancy. In the course of early placentation, trophoblast precursors differentiate into highly invasive trophoblast subtypes. These cells, referred to as extravillous trophoblasts (EVTs), penetrate the maternal uterus reaching as far as the inner third of the myometrium. One of the most fundamental functions of EVTs is the transformation of spiral arteries to establish the uteroplacental blood circulation assuring an adequate nutrient and gas supply to the developing fetus. To achieve this, specialized EVT subpopulations interact with maternal immune cells, provoke elastolysis in the arterial wall and replace the endothelial cells lining the spiral arteries to induce intraluminal vascular remodeling. These and other trophoblast-mediated processes are tightly controlled by paracrine signals from the maternal decidua and furthermore underlie an intrinsic cell-type specific program. Various severe pregnancy complications such as preeclampsia or intrauterine growth retardation are associated with abnormal EVT function, shallow invasion, and decreased blood flow to the placenta. Hence a better understanding of human trophoblast invasion seems mandatory to improve therapeutic intervention. This approach, however, requires a profound knowledge of the human placenta, its various trophoblast subtypes and in particular a better understanding of the regulatory network that controls the invasive phenotype of EVTs.     

Atrial natriuretic peptide binding in rat placenta,yolk sac,decidua, and materal placental vessels

Using in vitro autoradiography, binding sites of ¹²⁵I-ANP (atrial natriuretic peptide) were localized in the rat placenta, visceral yolk sac, and decidua at 16, 18, and 20 days of gestation. There was diffuse binding over the labyrinthine region of the placenta and an intense binding over the decidual gland and visceral yolk sac. In the yolk sac, ANP localized over the cores of the villi where it may be involved with the regulation of transport across the membranes or the flow of blood through the vitelline vessels. Of particular interest was binding over the maternal blood vessels supplying the decidual region and placenta. Receptors were located on the endothelial cells and smooth muscle cells of the arteries and veins, indicating that ANP may be involved with regional regulation of blood flow to the placenta.     

CCN1 (CYR61) and CCN3 (NOV) signaling drives human trophoblast cells into senescence and stimulates migration properties

ABSTRACT

During placental development, continuous invasion of trophoblasts into the maternal compartment depends on the support of proliferating extravillous trophoblasts (EVTs). Unlike tumor cells, EVTs escape from the cell cycle before invasion into the decidua and spiral arteries. This study focused on the regulation properties of glycosylated and non-glycosylated matricellular CCN1 and CCN3, primarily for proliferation control in the benign SGHPL-5 trophoblast cell line, which originates from the first-trimester placenta. Treating SGHPL-5 trophoblast cells with the glycosylated forms of recombinant CCN1 and CCN3 decreased cell proliferation by bringing about G0/G1 cell cycle arrest, which was accompanied by the upregulation of activated Notch-1 and its target gene p21. Interestingly, both CCN proteins increased senescence-associated β-galactosidase activity and the expression of the senescence marker p16. The migration capability of SGHPL-5 cells was mostly enhanced in response to CCN1 and CCN3, by the activation of FAK and Akt kinase but not by the activation of ERK1/2. In summary, both CCN proteins play a key role in regulating trophoblast cell differentiation by inducing senescence and enhancing migration properties. Reduced levels of CCN1 and CCN3, as found in early-onset preeclampsia, could contribute to a shift from invasive to proliferative EVTs and may explain their shallow invasion properties in this disease.     

Chorioallantoic and yolk sac placentation in Thrichomys laurentinus (Echimyidae) and the evolution of hystricognath rodents

The evolutionary history of Hystricognathi is associated with major transformations in their placental system. Data so far indicate that key characters are independent from size dimensions in medium to very large species. To better understand the situation in smaller species, we analyzed placental development in a spiny rat, Thrichomys laurentinus. Fourteen individuals ranging from early implantation to near term were investigated by histology, immunohistochemistry, proliferation activity and electron microscopy. Placentation in Thrichomys revealed major parallels to the guinea pig and other hystricognath rodents with respect to the early and invasive implantation, the process of trophoblast invasion, the internal organization of the labyrinth and the trophospongium as well as the establishment of the complete inverted yolk sac placenta. In contrast to systematically related small-sized species, the placental regionalization in Thrichomys was characterized by a remarkable lobulated structure and associated growing processes. Reverse to former perspectives, these conditions represented ancient character states of hystricognaths. The subplacenta was temporarily supplied by both the maternal and fetal blood systems, a rare condition among hystricognaths. The extraplacental trophoblast originating from the subplacenta was partly proliferative in mid gestation. In conclusion, the presented results indicated that only minor variations occurred in small-sized hystricognath species, independent of their systematic interrelationships. Previous views were supported that placentation in hystricognaths followed an extraordinary stable pattern, although the group had distinct habitats in South America and Africa that were separated 30-40 million years ago.     

Placentation in watersnakes I: Placental histology and development in North American Nerodia (Colubridae: Natricinae)

As part of a broad survey of placental structure, function, and evolution in reptilian sauropsids paraffin‐section histology was used to study microscopic anatomy of the uterus and fetal membranes of three species of North American watersnakes (Nerodia : Colubridae). The pre‐ovulatory uterus is poorly vascularized with inactive shell glands. These shell glands are activated during vitellogenesis but regress during pregnancy. Two placentas develop through apposition of the uterine lining to the chorioallantois and the yolk sac omphalopleure. Fetal and maternal components of the chorioallantoic placenta are progressively vascularized during development. Their epithelia are attenuated, but (contrary to a previous report), epithelia of neither the uterus nor the chorion are eroded. The fetal portion of the yolk sac placenta is an omphalallantois, formed of avascular omphalopleure, isolated yolk mass, and allantois. This placenta is progressively replaced by chorioallantoic placenta during mid‐ to late‐development through depletion of the isolated yolk mass. The chorioallantoic placenta is anatomically specialized for maternal–fetal gas exchange, and its expansion during development reflects the growing needs of the fetus for gas exchange. The yolk sac placenta is morphologically unsuited for gas exchange, but may serve other functions in maternal‐fetal exchange.     

Genetic insights into trophoblast differentiation and placental morphogenesis

The placenta is comprised of an inner vascular network covered by an outer epithelium, called trophoblast, all designed to promote the delivery of nutrients to the fetus. Several specialized trophoblast cell subtypes arise during development to promote this function, including cells that invade the uterus to promote maternal blood flow to the implantation site, and other cells that fuse into a syncytium, expand and fold to increase the surface area for efficient transport. Mutation of many genes in mice results in embryonic mortality or fetal growth restriction due to defects in placental development. Several important principles about placental development have emerged from these studies. First, distinct molecular pathways regulate the differentiation of the various trophoblast cell subtypes. Second, trophoblast proliferation, differentiation and morphogenesis are highly regulated by interactions with adjacent cell types. Finally, the specific classes of mutant phenotypes observed in the placenta of knockout mice resemble those seen in humans that are associated with preeclampsia and intrauterine growth restriction.     

Placentation in watersnakes II: Placental ultrastructure in Nerodia erythrogaster (Colubridae: Natricinae)

Ultrastructure of the placental tissues from redbelly watersnakes (Nerodia erythrogaster ) was analyzed during late pregnancy to provide insight into placental development and function. Examination of the chorioallantoic placenta with transmission electron microscopy reveals that chorionic and uterine epithelia are extremely attenuated but intact and that the eggshell membrane is vestigial and lacks a calcareous layer. These features minimize the interhemal diffusion distance across the placenta. Scanning electron microscopy reveals that fetal and maternal components of the placentas are richly vascularized by dense networks of capillaries. Although the yolk sac omphalopleure has largely been replaced by chorioallantois by late gestation, it retains patches of yolk droplets and regions of absorptive cells with microvilli and abundant mitochondria. Transmission electron microscopy reveals that yolk material is taken up for digestion by endodermal cells. As yolk is removed, allantoic capillaries invade to occupy positions just beneath the epithelium, forming regions of chorioallantoic placentation. Ultrastructural features indicate that the chorioallantoic placenta is specialized for gas exchange, while the omphalallantoic (“yolk sac”) placenta shows evidence of functions in yolk digestion and maternal‐fetal nutrient transfer. Placental features of this species are consistent with those of other thamnophines, and are evolutionarily convergent on snakes of other viviparous clades.     

Irisin induces trophoblast differentiation via AMPK activation in the human placenta

Irisin, an adipokine, regulates differentiation and phenotype in various cell types including myocytes, adipocytes, and osteoblasts. Circulating irisin concentration increases throughout human pregnancy. In pregnancy disorders such as preeclampsia and gestational diabetes mellitus, circulating irisin levels are reduced compared to healthy controls. To date, there are no data on the role and molecular function of irisin in the human placenta or its contribution to pathophysiology. Aberrant trophoblast differentiation is involved in the pathophysiology of preeclampsia. The current study aimed to assess the molecular effects of irisin on trophoblast differentiation and function. First-trimester placental explants were cultured and treated with low (10 nM) and high (50 nM) physiological doses of irisin. Treatment with irisin dose-dependently increased both in vitro placental outgrowth (on Matrigel™) and trophoblast cell-cell fusion. Adenosine monophosphate-activated protein kinase (AMPK) signaling, an important regulator of cellular energy homeostasis that is involved in trophoblast differentiation and pathology, was subsequently investigated. Here, irisin exposure induced placental AMPK activation. To determine the effects of irisin on trophoblast differentiation, two trophoblast-like cell lines, HTR-8/SVneo and BeWo, were treated with irisin and/or a specific AMPK inhibitor (Compound C). Irisin-induced AMPK phosphorylation in HTR-8/SVneo cells. Additionally, as part of the differentiation process, integrin switching from α6 to α1 occurred as well as increased invasiveness. Overall, irisin promoted differentiation in villous and extravillous cell-based models via AMPK pathway activation. These findings provide evidence that exposure to irisin promotes differentiation and improves trophoblast functions in the human placenta that are affected in abnormal placentation.     

Cytological distribution of chorionic gonadotropin subunit and placental lactogen messenger RNA in neoplasms derived from human placenta

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotropin (hCG), and placental lactogen (hPL). There are several gestational trophoblastic diseases of the placenta called hydatidiform mole, invasive mole, and choriocarcinoma. Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and small quantities of hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their messenger RNA (mRNA) in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha and - beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and -beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and -beta in the syncytial- like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues that contain cytotrophoblastic elements.     

The chemokines, CX3CL1, CCL14, and CCL4, promote human trophoblast migration at the feto-maternal interface

Human embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. Chemokines, critical regulators of leukocyte migration, are abundant in endometrial epithelial and decidual cells at this time. We hypothesized that endometrial chemokines stimulate trophoblast invasion. Chemokine receptors CX3CR1 and CCR1 were immunolocalized in human first-trimester implantation sites, specifically to endovascular extravillous trophoblasts, but not to the invading interstitial EVTs (iEVTs), with weak staining also on syncytium. CCR3 was localized to invading iEVTs and to microvilli on the syncytial surface. Expression of CX3CL1 (fractalkine), CCL7 (MCP-3), and their receptors (CX3CR1, CCR1, CCR2, CCR3, and CCR5) mRNA was examined in cellular components of the maternal-embryonic interface by RT-PCR. Both chemokines were abundant in entire endometrium and placenta, endometrial cells (primary cultures and HES, a human endometrial epithelial cell line) and trophoblast cell lines (JEG-3, ACIM-88, and ACIM-32). Chemokine receptor mRNA was expressed by placenta and trophoblast cell lines: CCR1 by all trophoblast cell types, whereas CCR2, CCR3, and CX3CR1 were more variable. CX3CR1, CCR1, CCR2, and CCR5 were also expressed by endometrial cells. Migration assays used the trophoblast cell line most closely resembling extravillous cytotrophoblast (AC1M-88). Trophoblast migration occurred in response to CX3CL1, CCL14, and CCL4, but not CCL7. Endometrial cell-conditioned media also stimulated trophoblast migration; this was attenuated by neutralizing antibodies to CX3CL1 and CCL4. Thus, chemokines are expressed by maternal and embryonic cells during implantation, whereas corresponding receptors are on trophoblast cells. Promotion of trophoblast migration by chemokines and endometrial cell conditioned medium indicates an important involvement of chemokines in maternal-fetal communication.     

Scanning electron microscopy of the placental interface in the viviparous lizard Sceloporus jarrovi (Squamata: Phrynosomatidae)

Placental membranes mediate maternal‐fetal exchange in all viviparous reptilian sauropsids. We used scanning electron microscopy to examine the placental interface in the mountain spiny lizard, Sceloporus jarrovi (Phrynosomatidae). From the late limb bud stage until birth, the conceptus is surrounded by placental membranes formed from the chorioallantois and yolk sac omphalopleure. The chorioallantois lies directly apposed to the uterine lining with no intervening shell membrane. Both fetal and maternal sides of the chorioallantoic placenta are lined by continuous layers of flattened epithelial cells that overlie dense capillary networks. The chorioallantoic placenta shows specializations that enhance respiratory exchange, as well as ultrastructural evidence of maternal secretion and fetal absorption. The yolk sac placenta contains enlarged fetal and maternal epithelia with specializations for histotrophic nutrient transfer. This placenta lacks intrinsic vascularity, although the vascular allantois lies against its inner face, contributing to an omphallantoic placenta. In a specialized region at the abembryonic pole, uterine and fetal tissues are separated by a compact mass of shed shell membrane, yolk droplets, and cellular debris. The omphalopleure in this region develops elongate folds that may contribute to sequestration and absorption of this material. Fetal membrane morphogenesis and composition in S. jarrovi are consistent with those of typical squamates. However, this species exhibits unusual placental specializations characteristic of highly placentotrophic lizards. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.