Different angioregulatory activity of monovalent galectin-9 isoforms.

Galectin-9 consists of two peptide-linked carbohydrate recognition domains (CRDs), but alternative splicing and proteolytic processing can give rise to multiple galectin-9 isoforms. Some of these consist of a single CRD and can exert different functions in cell biology. Here, we explored the role of these galectin-9 isoforms in endothelial cell function and angiogenesis. For this, we compared the effects of the two separate CRDs (Gal-9N and Gal-9C) with the tandem repeat galectin-9M on endothelial cell proliferation, migration, sprouting and tube formation in vitro as well as on angiogenesis in vivo using the chicken chorioallantoic membrane (CAM) assay. Galectin-9 isoforms significantly affected proliferation in quiescent endothelial cells and migration in activated endothelial cells. Interestingly, both monovalent gal-9 CRDs displayed opposite effects compared to gal-9M on proliferation and migration. Sprouting was significantly inhibited by gal-9C, while all isoforms appeared to stimulate tube formation. Angiogenesis in vivo was hampered by all three isoforms with predominant effects on vessel length. In general, the isoforms induced only subtle concentration-dependent effects in vitro as well as in vivo. Collectively, the effects of different galectin-9 isoforms in endothelial cell biology depend on the cellular activation status. While opposing effects can be observed on a cellular level in vitro, all galectin-9 isoforms hamper angiogenesis in vivo. This warrants further investigation of the regulatory mechanisms that underlie the diverging roles of galectin-9 isoforms in endothelial cell biology since this could provide therapeutic opportunities.

Application of the chick embryo chorioallantoic membrane in neurosurgery disease.

The chick embryo chorioallantoic membrane (CAM) is a highly vascularized extraembryonic membrane. Because of its ease of accessibility, extensive vascularization and immunodeficient environment, the CAM has been broadly used in the oncology, biology, pharmacy, and tissue regeneration research. The present review summarizes the application of the CAM in neurosurgery disease research. We focused on the use of the CAM as an assay for the research of glioma, vascular anomalies, Moyamoya Disease, and the blood-brain barrier.

Towards an understanding of the evolution of the chorioallantoic placenta: steroid biosynthesis and steroid hormone signaling in the chorioallantoic membrane of an oviparous reptile.

Amniotes, mammals, reptiles, and birds form common extraembryonic membranes during development to perform essential functions, such as protection, nutrient transfer, gas exchange, and waste removal. Together with the maternal uterus, extraembryonic membranes of viviparous (live-bearing) amniotes develop as an endocrine placenta that synthesizes and responds to steroid hormones critical for development. The ability of these membranes to synthesize and respond to steroid hormone signaling has traditionally been considered an innovation of placental amniotes. However, our laboratory recently demonstrated that this ability extends to the chorioallantoic membrane (CAM) of an oviparous (egg-laying) amniote, the domestic chicken, and we hypothesized that steroidogenic extraembryonic membranes could be an evolutionarily conserved characteristic of all amniotes because of similarities in basic structure, function, and shared evolutionary ancestry. In this study, we examined steroid hormone synthesis and signaling in the CAM of another oviparous amniote, the American alligator (Alligator mississippiensis). We quantified mRNA expression of a steroidogenic factor involved in the regulation of steroidogenesis (NR5A1), the key steroidogenic enzymes involved in the synthesis of progestins (HSD3B1), androgens (CYP17A1), and estrogens (CYP19A1), and the receptors involved in the signaling of progestins (PR), androgens (AR), estrogens (ESR1 and ESR2), and glucocorticoids (GR). Furthermore, we performed protein immunolocalization for PR and ESR1. Collectively, our findings indicate that the alligator CAM has the capability to regulate, synthesize, and respond to steroid hormone signaling, thus, supporting our hypothesis that the extraembryonic membranes of Amniota share a unifying characteristic, that is, the ability to synthesize and respond to steroid hormones.

Morphology and ultrastructure of the chorioallantoic placenta of the Iberian mole (Talpa occidentalis) with special reference to heterophagous areolas and the nature of interhaemal barrier.

This study provides a contribution to the reconstruction of the eulipotyphlan placental morphotype and also may help resolving a long-standing conflict about the interhaemal barrier in moles. As detailed descriptions of talpid placentation, only available for Talpa europaea and Scalopus aquaticus, led to a controversial debate on the nature of interhaemal barrier, the collection of more placental data of further mole species was strongly desired. Hence, the placentas of six gestational stages of Talpa occidentalis have been studied concerning their morphogenesis and ultrastructure with special focus on the structure of the interhaemal barrier and heterophagous regions. Generally, the mode of placentation in T. occidentalis resembles that of T. europaea, including a broad, discoid, antimesometrial, definitive chorioallantoic placenta of labyrinthine type being still villous in earlier stages. Within the labyrinth, the zona intima shows an endotheliochorial interhaemal barrier with a two-layered trophoblast. This clearly contradicts former statements on the S. aquaticus placenta made by Prasad et al. (1979), although their findings cannot exclude a totally different interpretation. Regardless, the placenta of moles represents the least invasive mode of placentation among Eulipotyphla, which otherwise have highly invasive placentas. Although the phagocytic areolas situated above uterine gland openings are heterophagous, they mainly seem to serve fetal histiotrophic nutrition, at least early in pregnancy. In later stages the number of glands and areolas decreases. This special type of additional phagocytic region is usually most common in species with noninvasive, epitheliochorial placentation, which suggests a correlation between placental invasiveness and the occurrence and type of phagocytic placental structures. The compact and invasive mode of placentation of Talpidae and all other Eulipotyphla seems to be plesiomorphic within Laurasiatheria and is always correlated with an altricial neonate.

The fetomaternal interface in the placenta of three species of armadillos (Eutheria, Xenarthra, Dasypodidae).

BACKGROUND: Placental characters vary among Xenarthra, one of four supraordinal clades of Eutheria. Armadillos are known for villous, haemochorial placentas similar to humans. Only the nine-banded armadillo has been well studied so far. METHODS: Placentas of three species of armadillos were investigated by means of histology, immunohistochemistry including proliferation marker, and transmission and scanning electron microscopy. RESULTS: The gross anatomy differed: Euphractus sexcinctus and Chaetophractus villosus had extended, zonary placentas, whereas Chaetophractus vellerosus had a disk. All taxa had complex villous areas within the maternal blood sinuses of the endometrium. Immunohistochemistry indicated the validity of former interpretations that the endothelium of the sinuses was largely intact. Tips of the villi and the columns entering the maternal tissue possessed trophoblast cell clusters with proliferation activity. Elsewhere, the feto-maternal barrier was syncytial haemochorial with fetal vessels near the surface. CONCLUSIONS: Differences among armadillos occurred in regard to the extension of the placenta, whereas the fine structure was similar. Parallels to the human suggest that armadillos are likely to be useful animal models for human placentation.

Morphology and development of the placentae in Eulamprus quoyii group skinks (Squamata: Scincidae).

Frequent evolutionary changes in reproductive mode have produced a wide range of placental structures in viviparous squamate reptiles. Closely related species with different placental structures and resolved phylogenetic relationships are particularly useful for reconstructing how placentae might have transformed during the evolutionary process. We used light microscopy to study placental morphology in mid- to late stage embryos of four closely related species of Eulamprus, a genus of viviparous scincid lizards that we had reason to suspect may display significant interspecific variation in placental morphology. Embryos from all four species possess a chorioallantoic placenta, an omphaloplacenta and an interomphalopleuric membrane, characteristics present in other viviparous skinks. However, unlike other viviparous skinks but characteristic of oviparous skinks, the allantois expands to surround the yolk sac in each species, supplanting the omphalopleure with a larger area of chorioallantois until a chorioallantoic placenta surrounds the entire egg in one specimen that is only a few days from birth. All four Eulamprus species share the same extraembryonic membrane morphology, but the cellular morphology of the uterine epithelium in the chorioallantoic placenta and omphaloplacenta varies between species. We determined that the interomphalopleuric membrane is a shared derived character of the Eulamprus quoyii species group. New phylogenetic information indicates that variation in the chorioallantoic placenta is a result of two independent transitions, but that variation in the omphaloplacenta can be explained using a single change within the species studied. Our results indicate that E. quoyii group skinks are a valuable model for investigating the evolution of viviparity, as extraembryonic membrane development in these species shows features characteristic of both oviparous and viviparous skinks.

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.

Optical coherence tomography and Doppler optical coherence tomography in the gastrointestinal tract.

Optical coherence tomography (OCT) is a noninvasive, high-resolution, high-potential imaging method that has recently been introduced into medical investigations. A growing number of studies have used this technique in the field of gastroenterology in order to assist classical analyses. Lately, 3D-imaging and Doppler capabilities have been developed in different configurations, which make this type of investigation more attractive. This paper reviews the principles and characteristics of OCT and Doppler-OCT in connection with analyses of the detection of normal and pathological structures, and with the possibility to investigate angiogenesis in the gastrointestinal tract.

Sexing of newly-hatched chicks using DNA isolated from chorio-allantoic membrane samples by polymerase chain reaction in Denizli chicken.

1. The aim of the present study was to determine the sex of newly-hatched chicks of Denizli chicken, a local Turkish breed, by polymerase chain reaction (PCR) using DNA extracted from the chorioallantoic membrane (CAM). 2. Fertilised eggs were incubated individually and a total of 20 CAM samples were collected following the hatching process. DNA was isolated from the CAM samples and PCR was performed using W-repeat (W) and 18 S ribosomal gene (R) primers. 3. Screening of the PCR products by agarose gel electrophoresis revealed that males have a single band (256 bp) and females have an extra second band (415 bp) as expected. 4. The present study describes a reliable, rapid, and simple multiplex PCR protocol that can be put into use to sex local breeds of chicken in which phenotypic sexing is impossible, using DNA isolated from the CAM that is discarded and remains attached to the egg shell following the hatching process.

Liver reconstruction on the chorioallantoic membrane of the chick embryo.

The liver from a 6-day-old chick embryo was transplanted on the chorioallantoic membrane of a 9-day-old chick embryo to observe the process of liver regeneration histologically. When a piece of the liver was implanted on the chorioallantoic membrane, only cells in the superficial zone of the graft adhering to the chorioallantoic membrane survived. Eventually, these surviving cells in the superficial zone proliferated with hematopoiesis, resulting in the formation of clusters of blood cells surrounded by the hepatocytes (or hepatic parenchymal cells). Semi-thin serial sections showed that these clusters of blood cells were confined to the space formed by hepatocytes. Furthermore, structures similar to the hepatic cord, sinusoid, central vein, and bile duct appeared in the reconstructed liver eleven days after transplantation. Meanwhile, when a pellet of the dissociated liver cells was transplanted onto the chorioallantoic membrane, two types of liver like structures were reconstructed: one was clusters of hepatocytes accompanied by sinusoids after hematopoiesis, and the other was a simple accumulation of hepatocytes without any sinusoids or hematopoiesis. The sinusoids found in the former type became clear following the connection between the space in the transplant and vessels of the chorioallantoic membrane. These findings indicated that the reconstructed liver was primarily produced by the accumulation of hepatocytes accompanied by hematopoietic cells, followed by the formation of sinusoidal spaces. We therefore consider that hematopoiesis is important for liver regeneration with a normal structure. Transplantation of the liver in the chorioallantoic membrane could be also useful for research into liver regeneration.

Evolução morfométrica dos anexos embrionários e fetais bovinos obtidos por monta natural, com 10 a 70 dias da gestação / Biometrics evolution of the embryonic and fetal annexes in cows obtained by natural mating, at 10 to 70 days of gestation

O período inicial da gestação de bovinos é caracterizado por grandes perdas embrionárias. Considerando a importância deste fator no âmbito da reprodução animal foram estudados os anexos embrionários e fetais bovinos fecundados por monta natural de 15-70 dias de gestação, com o objetivo de estabelecer parâmetros morfométricos da placenta na fase inicial da gestação. Com uso de um paquímetro foram realizadas mensurações do comprimento (crânio caudal), largura (latero lateral) e altura (dorso ventral) das membranas corioalantóide e amniótica. O início da formação dos cotilédones foi observado e quantificado, assim como, o peso placentário. O peso médio do saco gestacional aumentou com o evoluir da idade gestacional, entretanto, o crescimento foi acelerado a partir de 20-30 dias de gestação. O comprimento crânio caudal e dorso ventral da membrana corioalantóide e do âmnio apresentaram crescimento lento e gradual com o evoluir dos períodos gestacionais analisados. Com 30-40 dias de gestação, os primeiro cotilédones já eram visualizados e contatos com facilidade na superfície coriônica. Os períodos de crescimento coincidiram com os maiores índices de perdas gestacionais em bovinos. Os parâmetros aqui analisados poderão servir para futuras investigações dos anexos embrionários de organismos manipulados em laboratório.

The main goal of this morphometrical study was to characterize the development of the extra-embryonic membranes of 15 to 70-day-old bovine embryos obtained by natural mating. With a millimeter paquimeter the cranio-caudal, latero-lateral and dorso-ventral measurements of chorion and amnion were determined. The development of the cotyledons and weight of the gestational sac were observed and quantified. The weight of the gestational sac increased during gestation; however, the growth was faster then 20 to 30 days. The cranial-caudal and dorsal-ventral length of the choriallantois and the amnion developed slowly and gradually with progress of the gestational period, and the cranio-caudal length decreased after 50 to 60 days of gestation, increasing in the next period. After 30 to 40 days of gestation, the first cotyledons were visualized and counted easily in the chorionic surface. The growth periods were the same of the main gestational losses in cattle. The parameters analyzed could be useful for investigations of extra-embryonic membranes in organisms manipulated in the laboratory.

Regional and developmental variations of blood vessel morphometry in the chick embryo chorioallantoic membrane.

Avian eggs contain all the necessary materials for embryonic development except for oxygen, which diffuses in from the environment via pores in the hard, calcified eggshell to the chorioallantoic membrane (CAM), the respiratory organ, which is rich in blood vessels. An air cell is formed at the blunt pole of the egg between the two membranes of the eggshell and enlarges during incubation due to water vapor loss. In this study of the CAM of chicken eggs, we compared blood vessel numerical density [N(A(v))], area fraction of blood vessels [A(A(v))], CAM thickness (D(CAM)), total length of blood vessels (L) and surface area of the CAM attached to the eggshell (CAMre) with those under the air cell (CAMac) during incubation. We found that N(A(v)), A(A(v)), D(CAM) and L of the CAM increase with embryonic age and development. The N(A(v)), A(A(v)) and L under the air cell were higher in relation to the rest of the CAM at all ages tested, while the D(CAM) under the air cell was always lower than around the rest of the egg. Since the eggshell over the air cell has a relatively greater porosity, and the respiratory gas exchange ratio there is higher than at other areas of the egg, there is a correlation between all the above morphometric data and the eggshell porosity. This suggests optimization of embryonic gas exchange in the chicken egg. We would like to propose that, during natural incubation, an increased gas diffusion under the air cell, together with increased blood vessel numerical density, may compensate for covering of the central part of the eggshell by the incubating parent.