Urea-based osmoregulation in the developing embryo of oviparous cartilaginous fish (Callorhinchus milii) : contribution of the extraembryonic yolk sac during the early developmental period

Marine cartilaginous fish retain a high concentration of urea to maintain the plasma slightly hyperosmotic to the surrounding seawater. In adult fish, urea is produced by hepatic and extrahepatic ornithine urea cycles (OUCs). However, little is known about the urea retention mechanism in developing cartilaginous fish embryos. In order to address the question as to the mechanism of urea-based osmoregulation in developing embryos, the present study examined the gene expression profiles of OUC enzymes in oviparous holocephalan elephant fish (Callorhinchus milii) embryos. We found that the yolk sac membrane (YSM) makes an important contribution to the ureosmotic strategy of the early embryonic period. The expression of OUC enzyme genes was detectable in the embryonic body from at least stage 28, and increased markedly during development to hatching, which is most probably due to growth of the liver. During the early developmental period, however, the expression of OUC enzyme genes was not prominent in the embryonic body. Meanwhile, we found that the mRNA expression of OUC enzymes was detected in the extra-embryonic YSM; the mRNA expression of cmcpsIII in the YSM was much higher than that in the embryonic body during stages 28-31. Significant levels of enzyme activity and the existence of mitochondrial-type cmgs1 transcripts in the YSM supported the mRNA findings. We also found that the cmcpsIII transcript is localized in the vascularized inner layer of the YSM. Taken together, our findings demonstrate for the first time that the YSM is involved in urea-based osmoregulation during the early to mid phase of development in oviparous cartilaginous fish.

Development of the inverted visceral yolk sac in three species of caviids (rodentia, caviomorpha, caviidae)

Guinea pig related rodents possess numerous derived placental characters. We attempt to identify diversity within the visceral yolk sac and its association with the chorioallantoic placenta in three species of caviids, two of them possessing a capsule formed by the decidua that covers the chorioallantoic placenta. The results verify that in early pregnancy all three species have in inverted yolk sac placenta. In advanced pregnancy the species differ: Galea spixii, as representative without a capsule, bear a yolk sac in apposition to the chorioallantoic placenta with signs of exchange activity until term. Galea is similar to other caviomorphs in this respect. In Dasyprocta leporina and Cuniculus paca, the representatives possessing a capsule, the yolk sac endoderm lacks signs of substance exchange. Evidently, the presence of a capsule prevents such ail interaction. The variations established here must be considered if animal models for human placentation are required which have restricted access to the chorioallantoic placenta from the outside. (C) 2008 Elsevier Ltd. All rights reserved.

Yolk sac remnant in a mini-pony foal

Few reports are available concerning yolk sac remnants in the equine species, with little information on histopathological findings. A stillborn mini-pony foal showed a fluid-filled mass, measuring about 10 cm in circumference, close to the umbilical cord. Microscopic examination better defined the structures seen in the cyst.

Influence of sex, age, and fasting on blood parameters and body, bursa, spleen and yolk sac weights of broiler chicks

The effects of water and feed fasting for 24, 48 and 72 hours post-hatching on blood parameters (mean corpuscular volume, MCV; red blood-cell, RBC; hematocrit, HCT; hemoglobin, HGB; plasma glucose, CGP; plasma total protein, PP, and differential leukocytes count), and on body, liver, spleen, bursa, and yolk sac weights were analyzed. Erythrogram data were obtained with a blood cell counter. Total plasma protein and plasma glucose were determined by using the Bradford method (1976) and a glucose PAP liquiform kit (Labtest, cat. n. 84), respectively. Specific leukocyte counts were carried out on blood smears stained with Rosenfeld solution. According to the obtained data, water and feed post-hatching fasting reduced MCV values, which also were lower in males than that in females. Fasting for 48 hours promoted an increase in PP, while fasting for 72 hours reduced HCT. Chicks submitted to fasting presented lower body weights as compared to fed chicks, but their liver weight did not increase between 48 and 72 hours of age. Fasting decreased spleen weight, but bursa and yolk sac weight were not affected. Data showed that female and male chicks react in a similar way to post-hatching fasting, which affects body weight, liver and spleen weight, and HCT and PP values. Moreover, 72 hours of fasting affected more intensely HCT and MCV values.

Characterization of yolk sac proteins of Bos indicus cattle embryos

The yolk sac is an embryonic membrane that is essential for the embryo's initial survival in many mammals. It also plays an important role in the production of proteins necessary for development. We studied proteins of the yolk sac in bovine embryos at up to 40 days of gestation. We examined the yolk sac of 17 bovine embryos at different gestational periods, measuring a-fetoprotein, alpha-1-antitrypsin, and transferrin. This experiment was carried out by Western blot technique, associated with electrophoresis on a 6% sodium dodecyl sulfate polyacrylamide gel. Mouse monoclonal antibody anti-human-alpha-fetoprotein, mouse antibody anti-human-transferrin and rabbit polyclonal anti-human-alpha-1-antitrypsin were used as primary antibodies, and conjugated peroxidase as a secondary antibody. We detected the three proteins in some of the yolk sac samples; however, the bands in some specimens (samples) were weak, maybe a result of poor antigen-antibody reaction, since the antibodies used in this study were not specific to bovine proteins. The fact that weak bands appeared might be due to a weak cross-reaction.

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. J. Exp. Zool. (Mol. Dev. Evol.) 318:13-25, 2012. (C) 2011 Wiley Periodicals, Inc.

Morphology and Involution of the Yolk Sac during Early Gestation Bovine (Bos indicus)

Background: The bovine yolk sac derives from visceral endoderm and its development occurs between days 18-23 of gestation. The study of this membrane is important for comparative data and has already been performed in rodents, sheep and in cattle, especially Bos taunts. In species Bos indicus the yolk sac has not quite been studied and is believed that there are morphological differences between these species. The yolk sac undergoes a process of involution and degeneration during embryonic development and none vestige of it is found in late gestation. The period in which occurs the involution of the yolk sac coincides with the period of increased pregnancy loss in cattle, and changes in the morphology of this membrane may indicate the reasons for such high loss rates. Thus, considering that the yolk sac is important for embryonic circulation and metabolic transmission, besides participating actively in the process of cattle placentation, this study aimed characterize morphologically the involution of the bovine yolk sac. Materials, Methods & Results: The early gestational period was determined between days 20 and 70 post-insemination (p.i), according to the exterior characteristics of embryo/fetus. For macroscopic analyzes the uterus was dissected to expose the fetal membranes and subsequently the embryo/fetus was photographed. The samples were fixed for light microscopy and transmission electron microscopy. The yolk sac that emerges from the ventral part of the embryo was prominent and composed by a central part with two thin peripheral projections of different lengths. The bovine yolk sac with about 9 cm on day 25 p. i. of pregnancy permanently decreased its total length during this study. Histologically, the yolk sac is composed of three cell layers: the mesothelium, the mesenchyme and the endoderm. In mesenchyme are found blood islets. In the endoderm are formed cells invaginations toward the mesenchyme originating small canaliculi. The ultrastructure of yolk cells presented many mitochondria, rough endoplasmic reticulum, vesicles, euchromatin and the presence of two nucleoli, Discussion: The real first blood circulation in the bovine is attached with the development of yolk sac, differently from other membranes, such as the corium, that does not present evidence of vascularization by the age of 20-30 days. The erythroblasts found in the yolk sac are related to vasculogenesis and the process of differentiation of blood cells during the erythropoiesis. It could be observed on the histology of the yolk sac, in embryos of 30-50 days old, the presence of canaliculi and small folds of the epithelium. The canaliculi collapse is associated with the degeneration of the endoderm wall of the yolk sac. The organelles present in the endoderm cells of the yolk sac are associated with the function of protein metabolism and in the exchange of substances between the mesenchyme and the mesothelium, For these findings, could be observed that the yolk sac epithelium is found active until the 50th day of gestation, and thereafter regresses. However, remnants of this membrane may be present until the 70th day, These features may represent a presence of an active chorionvitelline placenta in this period responsible for the maintenance of pregnancy whereas the chorioallantoic placenta is not definitively established.

Development of of yolk sac inversion in Galea spixii and Cavia porcellus (Rodentia, Caviidae)

Caviomorph development includes an inverted yolk sac. Since principle processes are not understood, we investigated its differentiation in Galen and re-examined material from the guinea pig. Galea showed the typical caviomorph conditions in blastocyst development and the nature of the definitive yolk sac, formed of the visceral layer that became villous, proliferative, vascularized and attached to the uterus and placenta. In contrast to what was known before, in both species parts of the parietal yolk sac and a yolk sac cavity were temporarily present. Data suggest that early yolk sac development in caviomorphs is more complex than thought before. (C) 2012 Elsevier Ltd. All rights reserved.

An unusual feature of yolk sac placentation in Necromys lasiurus (Rodentia, Cricetidae, Sigmodontinae)

We studied the development of the inverted yolk sac in a New World rodent, Necromys lasiurus during early placentation. Ten implantation sites were investigated by means of histology, immunohistochemistry and electron microscopy. The yolk sac was villous near its attachment to the placenta. Elsewhere it was non-villous and closely attached to the uterus. The uterine glands were shallow and wide mouthed. They were associated with vessels and filled with secretion, suggesting the release of histotroph. This feature was absent at later stages. The intimate association of the yolk sac with specialized glandular regions of the uterus may represent a derived character condition of Necromys and/or sigmodont rodents. (C) 2012 Elsevier Ltd. All rights reserved.

In vitro and in vivo differentiation into B cells, T cells, and myeloid cells of primitive yolk sac hematopoietic precursor cells expanded >100-fold by coculture with a clonal yolk sac endothelial cell line

The yolk sac, first site of hematopoiesis during mammalian development, contains not only hematopoietic stem cells but also the earliest precursors of endothelial cells. We have previously shown that a nonadherent yolk sac cell population (WGA+, density <1.077, AA4.1+) can give rise to B cells, T cells, and myeloid cells both in vitro and in vivo. We now report on the ability of a yolk sac-derived cloned endothelial cell line (C166) to provide a suitable microenvironment for expansion of these early precursor cells. Single day 10 embryonic mouse yolk sac hematopoietic stem cells were expanded >100 fold within 8 days by coculture with irradiated C166 cells. Colony-forming ability was retained for at least three passages in vitro, with retention of the ability to differentiate into T-cell, B-cell, and myeloid lineages. Stem cell properties were maintained by a significant fraction of nonadherent cells in the third passage, although these stem cells expressed a somewhat more mature cell surface phenotype than the initial yolk sac stem cells. When reintroduced into adult allogeneic immunocompromised (scid) hosts, they were able to give rise to all of the leukocyte lineages, including T cells, B cells, and myeloid cells. We conclude that yolk sac endothelial cells can support the stable proliferation of multipotential hematopoietic stem cells, thus generating adequate numbers of cells for study of the mechanisms involved in their subsequent development and differentiation, for in vivo hematopoietic restitution, and for potential use as a vehicle for gene transfer.

Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis.

Presomitic and 3- to 12-somite pair cultured mouse embryos were deprived of retinoic acid (RA) by yolk-sac injections of antisense oligodeoxynucleotides for retinol binding protein (RBP). Inhibition of yolk-sac RBP synthesis was verified by immunohistochemistry, and the loss of activity of a lacZ-coupled RA-sensitive promoter demonstrated that embryos rapidly became RA-deficient. This deficiency resulted in malformations of the vitelline vessels, cranial neural tube, and eye, depending upon the stage of embryonic development at the time of antisense injection. Addition of RA to the culture medium at the time of antisense injection restored normal development implicating the role of RBP in embryonic RA synthesis. Furthermore, the induced RA deficiency resulted in early down-regulation of developmentally important genes including TGF-beta1 and Shh.

Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene.

The scl gene encodes a basic-helix-loop-helix transcription factor which was identified through its involvement in chromosomal translocations in T-cell leukemia. To elucidate its physiological role, scl was targeted in embryonic stem cells. Mice heterozygous for the scl null mutation were intercrossed and their offspring were genotyped. Homozygous mutant (scl-/-) pups were not detected in newborn litters, and analysis at earlier time points demonstrated that scl-/- embryos were dying around embryonic day 9.5. The scl-/- embryos were pale, edematous, and markedly growth retarded after embryonic day 8.75. Histological studies showed complete absence of recognizable hematopoiesis in the yolk sac of these embryos. Early organogenesis appeared to be otherwise normal. Culture of yolk sac cells of wild-type, heterozygous, and homozygous littermates confirmed the absence of hematopoietic cells in scl-/- yolk sacs. Reverse transcription PCR was used to examine the transcripts of several genes implicated in early hematopoiesis. Transcripts of GATA-1 and PU.1 transcription factors were absent from RNA from scl-/- yolk sacs and embryos. These results implicate scl as a crucial regulator of early hematopoiesis.