Capture of a Hyena-Specific Retroviral Envelope Gene with Placental Expression Associated in Evolution with the Unique Emergence among Carnivorans of Hemochorial Placentation in Hyaenidae.

Capture of retroviral envelope genes from endogenous retroviruses has played a role in the evolution of mammals, with evidence for the involvement of these genes in the formation of the maternofetal interface of the placenta. It has been shown that the diversity of captured genes is likely to be responsible for the diversity of placental structures, ranging from poorly invasive (epitheliochorial) to highly invasive (hemochorial), with an intermediate state (endotheliochorial) as found in carnivorans. The latter recapitulate part of this evolution, with the hyena being the sole carnivoran with a hemochorial placenta. In this study, we performed RNA sequencing on hyena placental transcripts and searched for endogenous retroviral envelope genes that have been captured specifically in the Hyaenidae clade and are not found in any other carnivoran. We identified an envelope gene that is expressed in the placenta at the level of the maternofetal interface, as evidenced by in situ hybridization/immunohistochemistry. The gene entry is coincidental with the emergence of the Hyaenidae clade 30 million years ago (Mya), being found at the same genomic locus in all 4 extant hyena species. Its coding sequence has further been maintained during all of Hyaenidae evolution. It is not found in any of the 30 other carnivorans-both Felidae and Canidae-that we screened. This envelope protein does not disclose any fusogenic activity in ex vivo assays, at variance with the syncytin-Car1 gene, which is found in all carnivorans, including the hyena, in which it is still present, transcriptionally active in the placenta, and fusogenic. Together, the present results illustrate the permanent renewal of placenta-specific genes by retroviral capture and de facto provide a candidate gene for the endotheliochorial to hemochorial transition of Hyaenidae among carnivorans.IMPORTANCE The placenta is the most diverse organ among mammals, due in part to stochastic capture of retroviral envelope genes. In carnivorans, capture of syncytin-Car1 took place 80 Mya. It is fusogenic, expressed at the syncytialized placental maternofetal interface, and conserved among all carnivorans, consistent with their shared endotheliochorial placenta. Hyenas are a remarkable exception, with a highly invasive hemochorial placenta, as found in humans, where disruption of maternal blood vessels results in maternal blood bathing the syncytial maternofetal interface. In this study, we identified a retroviral envelope gene capture and exaptation that took place about 30 Mya and is coincident with the emergence of the Hyaenidae, being conserved in all extant hyena species. It is expressed at the maternofetal interface in addition to the shared syncytin-Car1 gene. This new env gene, not present in any other carnivoran, is a likely candidate to be responsible for the specific structure of the hyena placenta.

Dynamic changes to claudins in the uterine epithelial cells of the marsupial Sminthopsis crassicaudata (Dasyuridae) during pregnancy.

The fluid that surrounds the embryo in the uterus contains important nourishing factors and secretions. To maintain the distinct microenvironment in the uterine lumen, the tight junctions between uterine epithelial cells are remodeled to decrease paracellular movement of molecules and solutes. Modifications to tight junctions between uterine epithelial cells is a common feature of pregnancy in eutherian mammals, regardless of placental type. Here we used immunofluorescence microscopy and western blot analysis to describe distributional changes to tight junctional proteins, claudin-1, -3, -4, and -5, in the uterine epithelial cells of a marsupial species, Sminthopsis crassicaudata. Immunofluorescence microscopy revealed claudin-1, -3, and -5 in the tight junctions of the uterine epithelium of S. crassicaudata during pregnancy. These specific claudins are associated with restricting passive movement of fluid between epithelial cells in eutherians. Hence, their function during pregnancy in S. crassicaudata may be to maintain the uterine luminal content surrounding developing embryos. Claudin-4 disappears from all uterine regions of S. crassicaudata at the time of implantation, in contrast with the distribution of this claudin in some eutherian mammals. We conclude that like eutherian mammals, distributional changes to claudins in the uterine epithelial cells of S. crassicaudata are necessary to support pregnancy. However, the combination of individual claudin isoforms in the tight junctions of the uterine epithelium of S. crassicaudata differs from that of eutherian mammals. Our findings suggest that the precise permeability of the paracellular pathway of the uterine epithelium is species-specific.

Lectin histochemistry reveals two cytotrophoblast differentiation pathways during placental development in the feline (Feliscatus).

INTRODUCTION: Placental glycosylation has been examined on eight feline placentae ranging from approximately 15 to 60 days post-conception as little is known about changes in glycan distribution in this species. METHODS: Specimens were resin embedded and lectin histochemistry was applied to semi-thin sections using a panel of 24 lectins and an avidin-biotin revealing system. RESULTS: Abundant tri-tetraantennary complex N-glycan and α-galactosyl residues found in the syncytium in early pregnancy were greatly reduced in mid-pregnancy, though retained at the invasion front in the syncytium (N-glycan) or cytotrophoblast layer (αGal). Some other glycans were also uniquely present in invading cells. Abundant polylactosamine was found in the infolding basal lamina of syncytiotrophoblast and the apical villous cytotrophoblast membrane. Syncytial secretory granules often clustered near the apical membrane abutting maternal vessels. Decidual cells selectively expressed ß-galactosyl residues throughout pregnancy and highly branched N-glycan levels increased over time. DISCUSSION: Glycan distribution changes significantly over pregnancy, probably relating to the development of transport and invasive properties of trophoblast which in the endotheliochorial placenta reaches the level of the maternal vessels. Highly branched complex N-glycans, often associated with invasive cells, N-Acetylgalactosamine and terminal α-galactosyl residues are present at the invasion front abutting the junctional zone of the endometrium. Abundant polylactosamine on the syncytiotrophoblast basal lamina may reflect the presence of specialised adhesive interactions, while clustering of glycosylated granules apically is probably associated with secretion and absorption of material via maternal vasculature. It is suggested that lamellar and invasive cytotrophoblast represent distinct differentiation pathways. 246 words.

Decidual cells and decidualization in the carnivoran endotheliochorial placenta.

Decidualization is considered a distinctive feature of eutherian pregnancy, and has appeared during evolution along with the development of invasive forms of placentation, as the endotheliochorial placenta. Although decidualization is not massive in carnivores, as it is in most species developing hemochorial placentas, isolated or grouped cells regarded as decidual have been documented and characterized, mainly in bitches and queens. For the majority of the remaining species of the order, data in the bibliography are fragmentary. In this article, general morphological aspects of decidual stromal cells (DSCs), their time of appearance and lasting, data about the expression of cytoskeletal proteins and molecules considered as markers of decidualization were reviewed. From the data reviewed, it follows that carnivoran DSCs take part either in the secretion of progesterone, prostaglandins, relaxin, among other substances, or at least in the signaling pathways triggered by them. Beyond their physiological roles, some of those molecules are already being used, or are yet under study, for the non-invasive endocrine monitoring and reproductive control of domestic and wild carnivores. Only insulin-like growth factor binding protein 1, among the main decidual markers, has been undoubtedly demonstrated in both species. Laminin, on the contrary, was found only in feline DSCs, and prolactin was preliminary reported in dogs and cats. Prolactin receptor, on the other hand, was found in both species. While canine DSCs are the only placental cell type expressing the nuclear progesterone receptor (PGR), that receptor has not been demonstrated neither in feline DSCs, nor in any other cell in the queen placenta, although the use of PGR blockers leads to abortion. Against this background, and from the data gathered so far, it is unquestionable that DSCs in carnivorans do play a pivotal role in placental development and health. The knowledge about placental physiology is critical for medical care and breeding management, primarily in domestic carnivores; it is also absolutely crucial for a conservation approach in the management of endangered carnivore species.

Endotheliochorial placental glycosylation reflects evolutionary divergence between Felidae species (Felis catus and Panthera leo) and Canidae (Canisfamiliaris).

Endotheliochorial cat (Felis catus) and lion (Panthera leo) term placentae and one 6 week placenta (term 60-63 days) from a dog (Canis familiaris) were stained with a panel of 24 lectins to compare glycosylation at the feto-maternal interface. Glycan expression in lion and cat placentae was very similar apart from the occurrence of terminal α-galactose in the lion trophoblast. The dog differed in several respects, particularly in the trophoblast, consistent with species-specific glycotypes differing according to the degree of their evolutionary divergence. The data suggest that evolutionary effects on the glycotype are most readily observed in trophoblast.

Comparative matrix metalloproteinase-2 and -9 expression and activity during endotheliochorial and hemochorial trophoblastic invasiveness.

To establish a functional placenta, its development needs adequate trophoblastic invasiveness. The intricate and complex morphological and molecular aspects regulating trophoblastic invasion during endotheliochorial placentation of domestic carnivores and their similarities and differences with the hemochorial placenta are still poorly understood. During placentation processes, from the time of implantation, trophoblast cells invade the uterine endometrium where they achieve extensive degradation and remodeling of extracellular matrix components; in this process, matrix metalloproteinases (MMPs), particularly MMP-2 and 9, have an essential role in rebuilding, cell migration, and invasiveness. This review provides an overview of comparative trophoblast invasive events and the expression and activity of MMP-2 and 9 during endotheliochorial and hemochorial placentation, emphasizing dog and mouse placental models. Understanding of trophoblastic invasiveness in two models of placentation, the intermediately invasive domestic carnivore endotheliochorial placenta, and the more highly invasive mouse hemochorial placenta, contributes to deepen knowledge of the trophoblast invasive processes and their diverse and complex human placental alterations, such as preeclampsia.

Proteomic profile of extracellular matrix from native and decellularized chorionic canine placenta.

Placental plasticity, employing rapid growth and remodeling to supply the growing fetus, is majorly related to its extracellular matrix (ECM) components. Thus, we studied the proteome profiled of canine native and decellularized placenta to characterize the proteome related to maintenance of a microenvironment and structure suitable for tissue engineering applications. Protein was profiled from native (n=3) and decellularized (n=3) 35-days old canine placenta using the mass spectrometer Orbitrap Fusion Lumos. A total of 52 proteins were filtered and revealed ontologies connected to skeleton structuration, collagen processing, germ layers formation, cell adhesion, response to amino acids, and others. Also, the major enriched pathways were ECM-receptor interaction, focal adhesion, PI3K-Akt signaling, protein digestion and absorption. Aside, proteins related to structure (collagens), cell adhesion (laminin and fibronectin), ECM remodeling (MMP2 and TIMP3) and vascularization (VEGF and RLN) were present in decellularized condition. Our findings support the requirement of a proteomic profile to visualize the maintenance of essential protein groups for ECM structuring and physiology, that should support functions related to cell adhesion, vasculogenesis and as a reservoir of soluble molecules. Altogether, the 35-days old decellularized canine placenta can provide an adequate microenvironment for cell anchoring for further regenerative medicine application.

Term placenta of the southern elephant seal (Mirounga leonina).

INTRODUCTION: The pinnipeds' placenta has been described as zonary, annular, labyrinthic and endotheliochorial, like that of the terrestrial carnivores. This article describes the placenta of Mirounga leonina, a phocid pinniped, focusing on some morphological features related to fetal nutrition. METHODS: Placental samples from three elephant seals were collected and conditioned after natural delivery at the Antarctic Specially Protected Area 132. Histological and ultrastructural studies were conducted; cytokeratins, vimentin, α-smooth muscle actin, and desmin proteins were detected using immunohistochemistry. RESULTS: The placentas were zonary, lobed, belt-shaped, and showed multiple vivid orange areas, which corresponded to bilirubin crystalline pigment found among chorionic villi and inside trophoblast cells. In the labyrinth, cytotrophoblast cells were isolated and there was a scant syncytium interposed between maternal and fetal vessels. Fetal vessels were small, round, and frequently intratrophoblastic, while maternal vessels were large, irregular, sinuous, and thin-walled. Vimentin and actin were detected in some scattered non-vascular cells throughout the labyrinth. Broad areas of degenerated and necrotic maternal components were also observed. DISCUSSION: The placentas of pinniped and fissiped carnivores share several traits. However, some remarkable features might maximize respiratory efficiency, collaborating to endure deep-diving hypoxia. Some of them, as the notably large sinuous maternal capillaries and fetal capillary indentation into the syncytium, are shared, e.g., by Phocidae and Mustelidae. Besides hemotropic nutrition taking place through an extremely narrow barrier, the abundant necrotic material and hematic products might allow substantial endocytosis of detritus even in term placentas, in this species giving birth to precocious offspring.

Comparative Placental Anatomy: Divergent Structures Serving a Common Purpose.

The placenta, one of the most important transient organs, forms by the apposition of fetal membranes and maternal tissues. Its role is to mediate physiological exchanges between mother and fetus. The word "apposition" covers a wide range of structural variations. It includes approximation, adhesion, interdigitation, or actual fusion between fetal and maternal tissues.1 Formation of the placenta establishes hemotropic nutrition for the fetus: essential metabolites must be provided to maintain the growing fetus, and these must come to it via the maternal circulatory system.2,3 Equally important, the placenta also provides oxygen and removes metabolic waste products from fetal blood. Nutritive and excretory roles of the placenta are not its only functions: it also has immune and endocrine activities.4 Nutrient and gas transport, waste removal, immunological protection of the fetus, and hormonal secretion influencing the maternal metabolism are all complex functions. They may also to some extent be conflicting purposes; hence, the placenta is a complex fetal organ. It is structurally adapted to perform its roles somewhat differently in different species, but the set of functions remain the same. Understandably, the placenta has been the subject of extensive research, and it will continue be an important topic thanks to its complexity. The intent of this chapter is to provide a simple description of placental anatomy using classic categories and to describe anatomical species variations in humans, important domestic animals, and the major laboratory species.

Differential Spatiotemporal Patterns of Galectin Expression are a Hallmark of Endotheliochorial Placentation.

PROBLEM: Galectins influence the progress of pregnancy by regulating key processes associated with embryo-maternal cross talk, including angiogenesis and placentation. Galectin family members exert multiple roles in the context of hemochorial and epitheliochorial placentation; however, the galectin prolife in endotheliochorial placenta remains to be investigated. METHOD OF STUDY: Here, we used immunohistochemistry to analyze galectin (gal)-1, gal-3 and gal-9 expression during early and late endotheliochorial placentation in two different species (dogs and cats). RESULTS: We found that during early feline gestation, all three galectin members were more strongly expressed on trophoblast and maternal vessels compared to the decidua. This was accompanied by an overall decrease of gal-1, gal-3 and gal-9 expressions in late feline gestation. In canine early pregnancy, we observed that gal-1 and gal-9 were expressed strongly in cytotrophoblast (CTB) cells compared to gal-3, and no galectin expression was observed in syncytiotrophoblast (STB) cells. Progression of canine gestation was accompanied by increased gal-1 and gal-3 expressions on STB cells, whereas gal-9 expression remained similar in CTB and STB. CONCLUSION: These data suggest that both the maternal and fetal compartments are characterized by a spatiotemporal regulation of galectin expression during endotheliochorial placentation. This strongly suggests the involvement of the galectin family in important developmental processes during gestation including immunemodulation, trophoblast invasion and angiogenesis. A conserved functional role for galectins during mammalian placental development emerges from these studies.

Early studies of placental ultrastructure by electron microscopy.

BACKGROUND: Transmission electron microscopy (TEM) was first applied to study placental ultrastructure in the 1950's. We review those early studies and mention the scientists that employed or encouraged the use of TEM. FINDINGS: Among the pioneers Edward W. Dempsey was a key figure who attracted many other scientists to Washington University in St. Louis. Work on human placental ultrastructure was initiated at Cambridge and Kyoto whilst domestic animals were initially studied by Björkman in Stockholm and electron micrographs of bat placenta were published by Wimsatt of Cornell University. CONCLUSIONS: Prior to the introduction of better fixation techniques, TEM images were of modest technical quality. Nevertheless they gave important insights into placental ultrastructure, particularly the nature of the maternal-fetal interface.