Search

Egg biochemical composition is among the main factors affecting offspring quality and survival during the yolk-sac stage, when larvae depend exclusively on yolk nutrients. These nutrients are primarily embedded in the developing oocytes during vitellogenesis. In aquaculture, assisted reproduction procedures may be applied enabling gamete production. For the European eel (Anguilla anguilla), reproductive treatment involves administration of pituitary extracts from carp (CPE) or salmon (SPE) to induce and sustain vitellogenesis. In the present study, we compared the influence of CPE and SPE treatments on offspring quality and composition as well as nutrient utilization during the yolk-sac stage. Thus, dry weight, proximal composition (total lipid, total protein), free amino acids, and fatty acids were assessed in eggs and larvae throughout the yolk-sac stage, where body and oil-droplet area were measured to estimate growth rate, oil-droplet utilization, and oil-droplet utilization efficiency. The results showed that CPE females spawned eggs with higher lipid and free amino acid contents. However, SPE females produced more buoyant eggs with higher fertilization rate as well as larger larvae with more energy reserves (estimated as oil-droplet area). Overall, general patterns of nutrient utilization were detected, such as the amount of total lipid and monounsaturated fatty acids decreasing from the egg stage and throughout the yolk-sac larval stage. On the contrary, essential fatty acids and free amino acids were retained. Notably, towards the end of the yolk-sac stage, the proximal composition and biometry of surviving larvae, from both treatments, were similar., (© 2022. The Author(s).)

Yolk-shell particles (YSPs) have attracted increasing attention from various research fields because of their low density, large surface area, and excellent loading capacity. However, the fabrication of polymer-based porous YSPs remains a great challenge. In this work, multifunctional polycaprolactone YSPs were produced using trineedle coaxial electrospraying with a simple nonsolvent process. TiO 2 -Ag nanoparticles and Ganoderma lucidum polysaccharides (GLPs) were encapsulated into the outer shell of the YSPs as the major antibacterial and antioxidant components, whereas iron oxide (Fe 3 O 4 ) nanoparticles were incorporated into the inner core to act as a photothermal agent. The morphology and structure, chemical composition, biocompatibility, antioxidant, and antibacterial effects of the fabricated YSPs, photothermal effects, and the release profile of the encapsulated GLP were studied in vitro. Furthermore, the in vivo wound healing effects of the YSPs and the laser-assisted therapy were explored based on a burn wound model on c57 mice.

A simple cell or tissue lysate can provide a sufficient quality and amount of template DNA for polymerase chain reaction (PCR). This protocol can be used to prepare embryonic tissues or yolk sac samples for PCR analysis, but it is also useful for adult tissues (such as tail) that do not lyse completely by boiling in SDS/NaOH. Nonionic detergents are substituted for SDS because they interfere less with the polymerase reaction., (© 2019 Cold Spring Harbor Laboratory Press.)

Often, genotyping of mouse embryos is required, and a small part, such as the yolk sac, can be used for this purpose. Here, DNA samples are prepared from extra-embryonic tissues by digestion with Proteinase K and subsequent extraction. The yolk sac of mid-gestation or later-stage embryos provides a sufficient amount of DNA for Southern analysis. Small tissues of a few hundred cells are used for genotyping early postimplantation- and preimplantation-stage embryos by polymerase chain reaction (PCR)., (© 2019 Cold Spring Harbor Laboratory Press.)

Two hundred turkey breeder hens and 24 viable toms of 30-35 weeks age of small white variety were distributed into two treatment groups having four replicates of 25 hens and three toms in each treatment. First four replicates were offered a turkey breeder diet (Diet A) (Nutrient requirements of poultry, 1994, National Academic Press, Washington, DC) and the rest four replicates were maintained on a higher plane of nutrition (Diet B) for 8-week duration. After 6 weeks of experimental feeding, two replicates from each treatment groups were vaccinated with ND (R 2 B) vaccine. Yolk sac of embryo from birds fed Diet B had a significantly higher (p < .05) IgG, IgM level and HI titre (log 2) than those fed Diet A. HI titre values of embryonic yolk sac from the vaccinated birds fed Diet B were significantly higher (p < .05) than that of the control groups. In addition, HI titre values were significantly higher (p < .05) in the day-old poults of the birds fed Diet B than that of those fed Diet A. There was significantly (p < .01) positive correlation between serum IgG and IgM of the breeder birds and day-old chicks. Similarly, there was significantly (p < .05) positive correlation between yolk IgG and IgM after 1-month experimental feeding and yolk sac IgG and IgM. Positive correlation (p < .05) also existed between yolk sac IgM and day-old chick serum IgM. Furthermore, the HI titres of breeder birds' serum at 14 days post-vaccination were positively correlated with their egg yolk after 10 and 15 days post-vaccination, yolk sac and day-old chicks. Thus, the study envisaged that a higher immunity in neonate poults from turkey breeders maintained on a higher plane of nutrition may be elicited as there was maternal transfer of antibodies from the serum of breeder birds to their offsprings through their yolk sac., (© 2018 Blackwell Verlag GmbH.)

Developing a nanoscale drug delivery system with magnetic resonance imaging (MRI)/fluorescence imaging (FL) visibility to optimize the delivery efficiency and therapeutic efficacy under image guidance has attracted great attentions in the area of nanomedicine. Herein, a novel permeable yolk-shell structured gadolinium-doped quantum dots nanocomposite was synthesized as a theranostic nanocarrier via an indirectly doping method. The as-prepared permeable nanoparticles with tunable color fluorescent emission, paramagnetic and accessible mesoporous channels could be developed as a novel nanomedical platform for integrated multimodal diagnosis and therapy. The hydrophilic nanocomposites exhibited tunable fluorescence as well as high longitudinal relaxivity (r 1 = 17.32mM -1 s -1 ) in water with good colloidal stability. In vivo animal experiments further verified CSSP could achieve FL/MRI dual modality imaging. The widely used antineoplastic anthracycline drug doxorubicin (DOX) was absorbed into the permeable nanospheres with 95.3% loading efficiency and released in a pH-sensitive pattern. In vitro cancer cell cytotoxicity tests verified that the DOX-loaded nanocomposites had enhanced cytotoxicity compared with free DOX at the same concentration. The as-prepared nanocomposites present great potential as MRI/FL-visible nanoscale drug carrier to realize imaging-guided personalized therapy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The zebrafish is a useful vertebrate model to study lipid metabolism. Oil Red-O (ORO) staining of zebrafish embryos, though sufficient for visualizing the localization of triglycerides, was previously inadequate to quantify neutral lipid abundance. For metabolic studies, it is crucial to be able to quantify lipids during embryogenesis. Currently no cost effective, rapid and reliable method exists to quantify the deposition of neutral lipids and triglycerides. Thin layer chromatography (TLC), gas chromatography and mass spectrometry can be used to accurately measure lipid levels, but are time consuming and costly in their use. Hence, we developed a rapid and reliable method to quantify neutral lipids and triglycerides. Zebrafish embryos were exposed to Rimonabant (Rimo) or WIN 55,212-2 mesylate (WIN), compounds previously shown to modify lipid content during zebrafish embryogenesis. Following this, ORO stain was extracted out of both the zebrafish body and yolk sac and optical density was measured to give an indication of neutral lipid and triglyceride accumulation. Embryos treated with 0.3 microM WIN resulted in increased lipid accumulation, whereas 3 microM Rimo caused a decrease in lipid accumulation during embryogenesis. TLC was performed on zebrafish bodies to validate the developed method. In addition, BODIPY free fatty acids were injected into zebrafish embryos to confirm quantification of changes in lipid content in the embryo. Previously, ORO was limited to qualitative assessment; now ORO can be used as a quantitative tool to directly determine changes in the levels of neutral lipids and triglycerides.

We examined the distribution of silver in pregnant mice and embryos/fetuses following intravenous injections of 10 nm silver nanoparticles (AgNPs) or soluble silver nitrate (AgNO3) at dose levels of 0 (citrate buffer control) or 66 µg Ag/mouse to pregnant mice on gestation days (GDs) 7, 8 and 9. Selected maternal tissues and all embryos/fetuses from control, AgNP- and AgNO3-treated groups on GD10 and control and AgNP-treated groups on GD16 were processed for the measurement of silver concentrations, intracellular AgNP localization, histopathology and gross examination of tissue morphology. Inductively-coupled plasma mass spectrometry revealed silver in all examined tissues following either AgNP or AgNO3 treatment, with highest concentrations of silver in maternal liver, spleen and visceral yolk sac (VYS), and lowest concentrations in embryos/fetuses. For VYS, mean silver concentration following AgNO3 treatment (4.87 ng Ag/mg tissue) was approximately two-fold that following AgNP treatment (2.31 ng Ag/mg tissue); for all other tissues examined, mean silver concentrations following either AgNP or AgNO3 treatment were not significantly different from each other (e.g. 2.57 or 2.84 ng Ag/mg tissue in maternal liver and 1.61 or 2.50 ng Ag/mg tissue in maternal spleen following AgNP or AgNO3 treatment, respectively). Hyperspectral imaging revealed AgNP aggregates in maternal liver, kidney, spleen and VYS from AgNP-treated mice, but not AgNO3-treated mice. Additionally, one or more embryos collected on GD10 from eight of ten AgNP-treated mice appeared small for their age (i.e. Theiler stage 13 [GD8.5] or younger). In the control group (N = 11), this effect was seen in embryos from only one mouse. In conclusion, intravenous injection of 10 nm AgNPs to pregnant mice resulted in notable silver accumulation in maternal liver, spleen and VYS, and may have affected embryonic growth. Silver accumulation in embryos/fetuses was negligible.

The role of lipids in providing energy and structural cellular components during vertebrate development is poorly understood. To elucidate these roles further, we visualized lipid deposition and examined expression of key lipid-regulating genes during zebrafish embryogenesis. We also conducted a semiquantitative analysis of lipidomic composition using liquid chromatography (LC)-mass spectrometry. Finally, we analyzed processing of boron-dipyrromethene (BODIPY) lipid analogs injected into the yolk using thin layer chromatography. Our data reveal that the most abundant lipids in the embryo are cholesterol, phosphatidylcholine, and triglyceride. Moreover, we demonstrate that lipids are processed within the yolk prior to mobilization to the embryonic body. Our data identify a metabolically active yolk and body resulting in a dynamic lipid composition. This provides a foundation for studying lipid biology during normal or pharmacologically compromised embryogenesis., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Broilers are known as an efficient source of lean meat. Genetic selection resulted in broiler strains with large body size and fast growth, but a concomitant increase in fat deposition also occurred. Other than reducing nutrient intake, there is a lack of alternative methods to control body fat composition of broilers. The present study assessed whether incubation temperature (machine temperatures: 36ºC, 37.5ºC, and 39ºC; eggshell temperatures: 37.4 ± 0.08°C, 37.8 ± 0.15ºC, and 38.8 ± 0.33°C, respectively.) from d 13 affects broiler hatchling fat deposition. We analyzed adipocyte hypertrophy and proliferation in 3 body regions; weight and chemical composition of yolk-free chicks and yolk sacs; and serum lipid profile. Increased incubation temperature reduced abdominal and cervical adipocyte size. Independently of temperature, cervical adipocytes were smaller and showed higher proliferation than adipocytes in the abdominal and thigh regions. Smaller cervical adipocytes were observed in birds from eggs incubated at 36ºC and 39ºC. With regard to weight and composition of chicks, ash content as a percentage of dry matter was the only variable affected by temperature; it was higher in chicks from eggs incubated at 36ºC than at 39ºC and showed no significant difference between chicks incubated at 39ºC and 37.5ºC. Absolute and relative weights of yolk sacs were higher from eggs incubated at 39ºC than at 36ºC, and these two treatments did not differ from the 37.5ºC control. Absolute measures of yolk sac lipids, moisture, dry matter, and crude protein content were lower in chicks from eggs incubated at 36ºC, and no significant differences were found for these variables between chicks from eggs incubated at 37.5ºC and 39ºC. Hatchlings from eggs incubated at 36°C had significantly higher cholesterol levels than chicks incubated at the other 2 temperatures, but no additional effects on blood lipids were detected. Incubation temperature manipulation during fetal development altered cervical and abdominal adipocyte size in broiler hatchlings and could become a tool in hatcheries to manipulate chick quality, although further studies are needed to evaluate its long-term effects., (© 2015 Poultry Science Association Inc.)

Egg shells are poultry industry byproducts with potential for use in various biological and agricultural applications. We have been interested in the membranes underlying the calcareous shell as a feed supplement, which showed potential to improve immunity and performance of post hatch poultry. Therefore, to determine their protein and peptide profiles, we extracted the egg shell membranes (ESM) from fresh unfertilized eggs with methanol and guanidine hydrochloride (GdHCl) to obtain soluble proteins for analysis by mass spectrometry. The methanol extract was subjected to matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), high-performance reverse phase liquid chromatographic separation (HPLC), and tandem mass spectrometry (MS/MS) to determine its peptide and protein profiles. The GdHCl extract was subjected to ESI-HPLC-MS/MS following trypsin digestion of reduced/alkylated proteins. Nine proteins from the methanol extract and >275 proteins from the GdHCl extract were tentatively identified. The results suggested the presence of several abundant proteins from egg whites, such as ovoalbumin, ovotransferrin, and lysozyme as well as many others associated with antimicrobial, biomechanical, cytoskeletal organizational, cell signaling, and enzyme activities. Collagens, keratin, agrin, and laminin were some of the structural proteins present in the ESM. The methanol-soluble fraction contained several clusterin peptides and defensins, particularly, two isoforms of gallin. The ratios of the two isoforms of gallin differed between the membranes obtained from brown and white eggs. The high abundance of several antimicrobial, immunomodulatory, and other bioactive proteins in the ESM along with its potential to entrap various microbes and antigens may make it a suitable vehicle for oral immunization of post hatch poultry and improve their disease resistance.

Elasmobranchs are a group of animals that typically occupy upper trophic levels in food webs and have a propensity to accumulate high contaminant concentrations. To date, few studies have investigated maternal offloading processes in sharks, despite the fact that this process represents a substantial source of exposure for young sharks and is a significant pathway for contaminant redistribution within marine ecosystems. Comparable to mammalian systems, scalloped hammerhead sharks (Sphyrna lewini) utilize a yolk-sac placental strategy to nourish young in utero, which may allow females to transfer contaminants to young. Organic contaminants (PCBs and chlorinated pesticides) were measured in livers of both females and males from several age classes that were collected from U.S. Atlantic waters, including two near-term pregnant females and their embryos. Adult female hammerheads (n = 3) were found to have lower levels of PCBs compared to the younger, adult male (mean ± SD, 11.1 ± 1.0 vs. 22.8 μg g(-1) lw), but had substantially higher concentrations of pesticides (4.1 ± 0.9 vs. 1.9 μg g(-1) lw). Embryos from the two litters (n = 36) had similar levels of summed organic contaminant concentrations (4.6 ± 0.9 μg g(-1) lw) and pregnant females were estimated to offload approximately 0.03-2.3% of their hepatic contaminant load to offspring. While the potential health impacts of these transferred contaminants is unknown, this is the first study to demonstrate that scalloped hammerheads are exposed to a substantial amount of contaminants prior to birth and document maternal offloading of organochlorines in a pseudo-placental shark species. Therefore, future research should continue to investigate the potential adverse effects these contaminants have on elasmobranch physiology.

Previous experiments have proved that thermodynamically unstable calcium carbonate vaterite can exist for long periods in the yolk sac of a pigeon embryo. The aim of this article was to demonstrate the effect of in vitro mineralization of yolk sac fluid on calcium carbonate by direct precipitation. Experiments were conducted using pigeon yolk sac fluid and using lecithin extracted from pigeon yolk sac fluid as a control to investigate the regulating effects of the organic components in the embryo on the formation of the calcium carbonate precipitate. Multiple characterization methods were employed to study the various morphological patterns, sizes, crystal growth, and crystal phase transformations of the calcium carbonate precipitates as regulated by the yolk sac fluid extracted at different stages of incubation. The experimental results demonstrate that as the incubation proceeds towards the later stages, the composition and environmental features of the yolk sac fluid become more favorable for the formation of relatively unstable calcium carbonate phases with high energies of the vaterite state. The experiments conducted with extracted lecithin as the template for crystal growth yielded similar results. A large amount of organic molecules with polar functional groups carried by the yolk sac fluid have strong effects and can both initially induce the crystallization and regulate the aggregation of calcium carbonate. Furthermore, this regulation process is found to be closely related to the lecithin contained in yolk sac fluid. These observations confirm the changes in yolk sac fluid composition during incubation have significant effects on the production of vaterite, which implicates the calcium transport during embryo growth., (© 2015 Poultry Science Association Inc.)

The reproductive success of precocial birds depends on investments in clutch formation and incubation. Egg quality strongly affects the phenotypic traits correlated with survival of the hatchling, but parental ability to maintain incubation temperature can also influence hatchling outcomes. The effect of incubation temperature on hatchling phenotype has been widely studied in reptiles but not in birds. The aim of this study was to explore the effects of egg mass and incubation temperature on the incubation period, hatchability, and hatchling phenotype of the mallard (Anas platyrhynchos). Mallard eggs were incubated under six constant incubation temperatures (ranging from 35.0° to 39.0°C). Hatchlings were weighed, and their structural size was measured. Some hatchlings were used for an examination of residual yolk sac mass and basic chemical composition of the yolk-free body. All investigated phenotypic traits except for chemical composition were positively correlated with egg mass. Incubation temperature did not affect hatchling body mass, but increased temperatures led to a decreased yolk-free body mass and structural size of hatchlings and to increased yolk sac mass. Our results suggest that there is a trade-off between the yolk-free body size and energetic reserves in the form of the yolk sac and that this trade-off is modulated by incubation temperature.

The objective of the present study was to examine the changes in yolk and yolk sac fatty acid composition and also to investigate egg content, yolk absorption, embryo development during incubation, and chick quality at hatch in eggs from 36- and 52-wk-old broiler breeders. The fatty acid profiles of the yolk, the yolk sac of embryos, and the residual yolk sac of chicks were analyzed before incubation, on d 18, and at hatch, respectively. Yolk sac weight, and embryo weight and length were measured on d 18, and chick weight and length were measured at hatch. Egg weight, yolk and albumen weight, yolk percentage, and yolk:albumen ratio increased as breeder age increased, but the albumen percentage decreased. Yolk absorption in absolute value (g) was higher in embryos from the old flock on d 18 and at hatch. Relative yolk absorption was similar between age groups on d 18, whereas it was higher in the young flock at hatch. Breeder age affected the yolk sac weight and was higher in the old flock during incubation. Embryo or chick weight and length, and yolk-free BW were affected by breeder age during incubation. These parameters were higher in the old flock with a difference of 3.7 g, 0.8 cm, and 2.6 g, respectively, on d 18 and 7.4 g, 1.4 cm, and 6.3 g, respectively, at hatch compared with the young flock. The effect of breeder age on fatty acid composition differed significantly by sampling day. Palmitic, stearic, oleic, and linoleic acids were major fatty acids in the fresh yolk, ranging from 13.02 to 29.24%. These were followed by palmitoleic and arachidonic acids ranging from 1.24 to 7.04%, with the remaining fatty acids below 1%. Higher concentrations of myristic, palmitoleic, and oleic acids and lower concentrations of heptadeconoic, stearic, linoleic, and arachidonic acids were found in the residual yolk sac of the young flock than the old flock. The results showed preferentially selective absorption of some fatty acids by the embryo during incubation., (© Poultry Science Association Inc.)

Calcium carbonate crystals are known to form in the yolk sacs of fertile pigeon eggs at late stages of incubation. The composition and structure of these crystals were investigated, the crystallization environment was inspected, and the physical chemistry constants of the yolk fluid were determined through the incubation period. Polarized light microscopy was used to observe the generation and distribution of calcium carbonate crystals in the yolk sac. In addition, X-ray diffraction was employed to analyze the composition and crystal phase of the yolk sac. A decalcification and deproteination method was established to analyze the ultrastructure and composition of the crystals, as well as the internal relationship between inorganic and organic phases of the crystals. Additionally, scanning electron microscopy, transmission electron microscopy, X-ray energy dispersive spectroscopy, and Fourier transform infrared spectroscopy were used to evaluate the characteristics of the crystals. Our results demonstrated that the calcium carbonate crystals were mainly composed of vaterite and calcite, with vaterite being the major component. Vaterite, a type of biomaterial generated by an organic template control, presented as a concentric hierarchical spherical structure. The organic nature of the biomaterial prevented vaterite from transforming into calcite, which is more thermodynamically stable than vaterite. Additionally, the configuration, size, and aggregation of vaterite were also mediated by the organic template. This bio-vaterite was found during the incubation period and is valuable in calcium transport during embryonic development., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Eggs (n = 1,800) obtained from Ross broiler breeders at 32 and 48 wk of age were incubated at either a constant temperature of 37.6°C throughout (T1), or the temperature was reduced for 6 h to 36.6°C each day during embryonic age (EA) 10 to 18 (T2). Yolk sac, liver, and brain fatty acid profiles and oxidant and antioxidant status of liver and brain were measured at EA 14, 19, and day of hatch (DOH). Fatty acid profiles of yolk sac, liver, and brain were influenced by age of breeder with significant breeder hen age × incubation temperature interactions. At EA 14, higher levels of 20:4n-6 and 22:6n-3 had been transferred from the yolk sac to T2 embryos from younger than older breeders, whereas for T1 and T2 embryos, yolk sac 20:4n-6 and 22.6n-3 values were similar for older breeders. Accumulation of 20:4n-6 and 22:6n-3 fatty acids in the liver of T1 and T2 embryos from younger breeders was similar; however, T2 embryos from older breeders had higher liver levels of 20:4n-6 and 22:6n-3 than T1 embryos. At EA 19, liver nitric oxide levels were higher for T2 embryos from younger breeders than those from breeders incubated at T1. Brain catalase levels of T2 embryos from younger breeders were higher than those from older breeders at DOH. Thus, changes in fatty acid profiles and catalase and nitric oxide production of brain and liver tissues resulting from 1°C lower incubation temperature from EA 10 to 18 reflect adaptive changes.

This study was performed to obtain information on yolk utilization in fasted and fed ostrich chicks posthatching. The fasted trial lasted for 7 d, whereas the fed trial continued for 16 d. Fasted ostrich chicks showed a decrease of 31.3 g of BW, with yolk weight decreasing by 28.9 g daily after hatching. Yolk weight comprised 28% of 1-d-old ostrich chick BW and decreased to 12% at 7 d of age. Only 44.4% of the fasted ostrich chick yolk was assimilated over the trial period. Crude protein content of the yolk decreased by 13.2 g daily. Fat content increased by 1.77% daily, whereas total yolk fat weight decreased with 8.91 g daily. Slaughter weight of fed ostrich chicks increased, with yolk weight decreasing by 16.3 g daily. Yolk content for fed ostrich chicks was 26% of BW at 2 d of age. Ostrich chicks absorb 30% of yolk over the first 4 d, 67% after 8 d, and only deplete the yolk after 14 d posthatch. Fasted ostrich chicks absorbed the yolk content at a rate of 28.9 g/d, compared with 22.3 g/d over the first 8 d and 16.3 g/d over the 16 d for fed ostrich chicks. The CP content of the yolk decreased by 6.84 g daily in fed ostrich chicks, whereas fat content of the yolk increased by 1.39% daily, although total yolk fat weight decreased by 6.61 g daily. Yolk weight and total CP decreased faster over the first 7 d in the fasted ostrich chicks compared with the fed ostrich chicks, which indicated that the decrease in yolk weight could be attributed to absorption of protein from the yolk. Fat content decreased faster over the first 8 d from the yolk of the fed ostrich chicks compared with that from the yolk of the fasted ostrich chicks, which could indicate that external feed has a positive influence on the absorption of fat from the yolk content.

Glycogen and glucose concentrations (mg/g of tissue) and amounts (mg) were determined in the yolks of fertile eggs on the day of set and in the yolk sac (YS) and liver of broiler chick embryos between 11 and 21 embryonic days of age (E). On the day of set, the yolk contained 50 mg of glucose (0.31% of yolk) but did not contain glycogen. During incubation, the amount of glucose in the YS increased from 20 mg on E11 to 60 mg on E19. A parallel increase in YS and liver glycogen concentrations (mg/g) during the last week of incubation implied a similar capacity for glycogen synthesis per gram of tissue. However, due to its larger size, the YS capacity for glycogen storage far exceeded that of the liver, which stored less than 12 mg of glycogen up to E19, as compared with more than 200 mg in the YS. Between E19 and 21, liver and YS glycogen amounts decreased by 10 mg and 100 mg, respectively. These results indicated that the YS is a glycogenic and perhaps gluconeogenic organ. We therefore evaluated the gene expression of glycogen synthase and glycogen phosphorylase as well as gluconeogenic enzymes (fructose 1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase) in the YS membrane and liver by real-time reverse-transcription PCR. Although the YS membrane and liver displayed different patterns of mRNA abundance, the high abundance of fructose 1,6-bisphosphatase mRNA in the YS membrane between E11 and 15, and the expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase, supported the postulated gluconeogenic abilities of the YS membrane and indicated its role in providing glucose to the embryo. Thus, glucose is probably synthesized in the YS, stored in the form of glycogen, and toward hatch, the YS may have the potential to transfer 10 times more glycogen-derived glucose to the embryo as compared with the liver. As such, the YS may play a major role in the synthesis and storage of glucose and its supply to the chick embryo toward hatch.

1. Increased concentrations of maternal corticosterone are deposited into egg yolk under stress conditions. This experiment investigated the effect of maternal corticosterone on yolk and yolk sac fatty acid profiles of eggs and developing broiler embryos. 2. At 42 weeks of age, 200 broiler breeder females were randomly divided into two groups: maternal corticosterone (MC): 2 mg/hen/d of corticosterone dissolved in 1 mL of 99% ethanol and mixed in the individual daily feed for a 14 d period, and control: 1 mL of ethanol added to the individual daily feed. Hens were inseminated, and eggs were collected daily from d 3 of corticosterone feeding to d 14 and incubated. Fatty acid profiles of egg yolk and yolk sac were analysed before incubation and at 12, 14, 18 and 21 d of incubation. 3. Yolk corticosterone concentrations were greater in MC eggs. Before incubation, eggs from MC had lower DHA (22 : 6 n-3) but higher stearic (18 : 0) and dihomo-gamma linolenic (20 : 3 n-6 Cis 8,11,14) acid content compared with the control. 4. Higher concentrations of stearic (18 : 0), elaidic (18 : 1 tr n-9), eicosenoic (20 : 1 n-9), eicosadienoic (20 : 2 n-6), dihomo-gamma linoleic (20 : 3 n-6) and arachidonic (20 : 4 n-6) acids and lower concentrations of myristic (14 : 0), palmitoleic (16 : 1 n-7), linoleic (18 : 2 n-6) and DHA (22 : 6 n-3) were found at different periods of incubation in the residual yolk sac of MC than from controls. 5. The results suggest that the preferentially selective absorption of fatty acids is related to specific tissue requirements.

Indium embryotoxicity was investigated by proteomic analysis with two-dimensional electrophoresis of rat embryos cultured from day 10.5 of gestation for 24h in the presence of 50 microM indium trichloride. In the embryo proper, indium increased quantity of several protein spots including those identified as serum albumin, phosphorylated cofilin 1, phosphorylated destrin and tyrosyl-tRNA synthetase. The increased serum albumin, derived from the culture medium composed of rat serum, may decrease the toxicity of indium. The increase of phosphorylated cofilin 1 might be involved in dysmorphogenicity of indium through perturbation of actin functions. In the yolk sac membrane, indium induced quantitative and qualitative changes in the protein spots. Proteins from appeared spots included stress proteins, and those from decreased or disappeared spots included serum proteins, glycolytic pathway enzymes and cytoskeletal proteins, indicating yolk sac dysfunction. Thus, several candidate proteins that might be involved in indium embryotoxicity were identified.

The objectives of this study were to examine the relationship between thiamine concentrations in unfertilized eggs and yolksac individuals of lake trout (Salvelinus namaycush), along with any associated histopathological changes in the tissues of alevins at the hatching stage. We address these questions in a lake trout population from different spawning grounds of Lake Michigan (North and South), known for compromised survival due to early mortality syndrome (EMS). However, a dichotomous forage base of lake trout spawning stocks, with a dietary thiaminase-rich alewife in the North, and dietary low-thiaminase round goby in the South, provides the basis for the assumption that different diets may lead to differences in severity of EMS between different stocks. Lake trout eggs of 18 females were collected and fertilized individually with the sperm of several males. The eggs, eyed embryos and newly-hatched alevins were sampled to examine thiamine utilization during embryogenesis. Progenies of females with low (< 0.73 nmol/g) and high (> 0.85 nmol/g) levels of thiamine were chosen for histological studies. The obtained results showed that total thiamine levels in the body and yolk of eyed embryos and alevins at hatching were influenced by thiamine levels of unfertilized eggs and it decreased during embryogenesis (to 51% in eyed embryos and 28% in newly-hatched alevins in comparison to unfertilized eggs). The survival of lake trout until hatching stage does not correlate with the thiamine level, however it was affected by collection site and was significantly higher in fish from the South site (Julian's Reef). At the hatching stage, no pathological changes were observed in the brain, olfactory lobe, retina or liver in embryos regardless of thiamine concentrations in unfertilized eggs. It has been concluded that an enhanced thiamine requirement for the fast muscle mass growth near the swim-up stage is responsible for overt and histopathological signs of EMS. Current study confirms earlier findings that lake trout suffering from EMS can be successfully treated by immersion in thiamine solution as late as at the swim-up stage.

Acute and chronic bioassays were used to evaluate the lethal and sublethal effects of copper chloride and tributyltin chloride on mahi mahi (Coryphaena hippurus) embryos, a pelagic life stage often found in the surface microlayer where anthropogenic contaminants can accumulate. Acute bioassay testing determined the median lethal concentration (LC50) for the test organism after 48 h of exposure. Chronic toxicity tests were used to determine the measurement of sublethal parameters, such as developmental abnormalities after 72-h exposures to the toxicants. Embryos were collected 4 h postfertilization and subsequently exposed to 1, 5, 25, 50, and 100 microg/L of copper chloride and 3, 10, 20, 30, and 50 microg/L of tributyltin chloride. Analysis of hatch rate percentage determined that the mean 48-h LC50 of copper chloride and tributyltin chloride was 32.8 and 16.7 microg/L, respectively, based on the pooled data of four experiments with four replicates for each metal. Consistent abnormalities, such as yolk sac swelling, spinal deformities, and decreased hatch rates, were observed for each metal. Teratogenic responses to copper chloride and tributyltin chloride demonstrate the need to investigate further the impacts of pollution in the open oceans to a species indigenous to, and commercially important to, the Florida (USA) coasts. This information could then lead to the future development of a surface microlayer bioassay using mahi mahi embryos.

A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16-17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F(2 alpha) (PGF(2 alpha)). Uterocalin and beta(2)-microglobulin (beta(2)M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were similar in conceptuses from control and treated mares. A 17 kDa cationic protein identified as a secretory phospholipase A2 (sPLA2) type IIA was detected bound to normal capsules but increased substantially in response to PGF(2 alpha). Two forms of uteroglobin were distinguished by partial amino acid sequences of approximately 6 kDa bands in flush fluids from normal pregnant uteri. After administration of PGF(2 alpha) one immunoreactive form of uteroglobin was preferentially increased. These studies demonstrate that failure of pregnancy in this model is associated with an increase in secretory phospholipase in the capsule and a change in the forms of uteroglobin in the uterine secretions.

Background: Developmental toxicity of selenium (Se) is a nutritional, environmental and medicinal concern. Here, we investigated Se embryotoxicity by proteomic analysis of cultured rat embryos., Methods: Rat embryos at day 9.5 or 10.5 of gestation were cultured for 48 or 24 h, respectively, in the presence of sodium selenate (100 or 150 microM) or sodium selenite (20 or 30 microM). Proteins from the embryo proper and yolk sac membrane were analyzed by two-dimensional electrophoresis for quantitative changes from those in control embryos. Proteins with quantitative changes were identified by mass spectrometric analysis., Results: Growth inhibition and morphological abnormalities of cultured embryos were observed in all the Se treatment groups. By the analysis of the embryo proper, actin-binding proteins were identified as proteins with quantitative changes by selenate: increased phosphorylated-cofilin 1, increased phosphorylated-destrin, decreased drebrin E, and decreased myosin light polypeptide 3. Many proteins showed similar changes between selenate and selenite, including increased ATP-synthase, decreased acidic ribosomal phosphoprotein P0, and decreased pyrroline-5-carboxylate reductase-like. In the yolk sac membrane, antioxidant proteins were identified for protein spots with quantitative changes by selenite: increased peroxiredoxin 1 and increased glutathione S-transferase., Conclusion: The identified proteins with quantitative changes by selenate or selenite were considered to be candidate proteins involved in Se embryotoxicity: the actin-binding proteins for selenate embryotoxicity, proteins with the similar changes for the common Se embryotoxicity and antioxidant proteins for modification of Se embryotoxicity by redox-related treatments. These proteins may also be used as biomarkers in developmental toxicity studies., ((c) 2008 Wiley-Liss, Inc.)

Expansion of the equine conceptus can be divided into blastocoel and yolk sac phases. The endodermal layer transforming the blastocoel into the yolk sac is completed around day 8 of pregnancy. From that time, the size of the spherical conceptus increases tremendously due mainly to the accumulation of fluid rather than cell multiplication. In this study, we have investigated the abundance and localisation of Na(+)/K(+)-ATPases and aquaporins (AQP) in the equine conceptus on days 8, 10, 12, 14 and 16 by multiplex reverse transcriptase PCR, Western blot and immunohistochemistry. During conceptus expansion, the ectoderm of the yolk sac exhibited basolateral abundance of alpha1ATPase, apical localisation of AQP5, and membrane and cytoplasmic expression of AQP3. With increasing conceptus size its cells showed an extensive enlargement of the apical membrane surface by microvilli. From day 14 onwards, the yolk sac endoderm forms arc-like structures with attaching sites to the ectodermal layer and shows intensive staining for alpha1ATPase, AQP5 and AQP3 in the membrane as well as in the cytoplasm. In the yolk sac ectoderm, the arrangement of these proteins is comparable with the collecting ducts of kidney with AQP2 being replaced by the closely related AQP5. The detection of phosphorylation sites for protein kinase A suggests a similar AQP5 traffic and regulation as known for AQP2 in the collecting ducts of the kidney. The arrangement of these proteins in equine embryos indicates at least partially the mechanism of conceptus expansion.

C(18) neutral steroid formation by cytochrome P450 aromatase has been recorded for several equine and porcine tissues. High activity of P450 aromatase is reflected in the quantities of estrogens in yolk-sac (y-s) fluid of early equine conceptuses. In a previous study of y-s fluid we detected large amounts of androgens by radioimmunoassay (RIA), using an antiserum for androstenedione (A(4)). Here, we report that RIA, following chromatography, gave tentative identification of the major peak as norandrostenedione (19-norA) not as A(4). Furthermore, even greater quantities of 19-norA seemed to be present in y-s fluid as a sulphoconjugate, as noted from extraction, solvolysis, HPLC, followed by RIA. Confirmation of these unusual findings was attained after further purification with two HPLC systems and definitive identification by LC-MS with an authentic standard of 19-norA. Initial extraction of the steroid sulphate as a methylene-blue complex also yielded 19-norA suggesting that the 3-enol form had enabled sulphoconjugation. The biological significance of retention mainly as a sulphate is not known; however, the large amounts of 19-norA found in the fluid accords well with reports on the catalytic activity shown in vitro by the blastocyst isozyme of P450 aromatase in the pig and horse.

Antinuclear antibodies, ANA, were usually detected with antigen of somatic cell nucleus. It has not been reported to detect ANA with egg cell nucleus as antigen. Enzyme linked immuosorbent assay, ELISA, coated with yolk was developed to detect ANA in our laboratory. A quality control test, cross absorption test, and cross antibody-induced test with yolk were performed. Results showed a good agreement between our method and IFA through measurement of the same samples from patients suspected of having rheumatic connective tissue diseases (Kappa=0.668, P=0.000). The results were not influenced by the RF and different sources of egg. CVs of inter-assay, were less than 10%. The cross absorption test was negative, as well; the ANA to somatic cell nucleus could be induced with egg cell nucleus. It is implied that there were both cross as well as overlapped Egg-ANA and Somatic-ANA. As egg nucleus, its volume was large, its purification was simple, so the better method might be established.

Individual differences in animal behavior can be attributed to genetic as well as non-genetic influences. One mechanism by which the behavioral phenotype of an individual can be shaped is via transmission of maternal sex steroids. In this study, we examined the role of yolk testosterone (T) in controlling neophobia in 9-month-old, sexually mature zebra finches (Taeniopygia guttata). Offspring hatched from either T-treated or control eggs were subjected to a sequential series of behavioral tests in which we measured the neophobic response and its persistence towards two unfamiliar stimuli. Birds from T-treated and control eggs did not differ in their latencies to approach and eat a novel food source during their first encounter. However, egg treatment affected subsequent habituation. Latencies decreased in both groups over a habituation period of 5 days, but considerably more so in T-offspring. Although males appeared to approach novel food faster than females, there was no overall sex effect during the habituation period. When a novel object was added in combination with the previously learned food stimulus, this caused an behavioral shift in approach latencies. In males, control offspring had significantly shorter latencies than T-offspring, whereas there was no difference among females. The latency to eat in the same test was not significantly affected by sex or egg treatment. Our results demonstrate long-term effects of prenatal T on neophobic responses in adult zebra finches. We hypothesize that prenatal T may be one underlying mechanism for individual differences routine formation.

The mRNA expression of GPRC5B, an orphan G protein-coupled receptor, is induced by retinoic acid (RA). Because RA plays critical roles in embryonic development, reproductive functions, metabolism and homeostasis, GPRC5B is also considered crucial in these physiological events. We investigated the changes in expression of GPRC5B and RA receptor (RAR) alpha mRNAs and immunohistochemical localization of their proteins in the murine placenta and yolk sac at 13.5, 15.5 and 17.5 days post coitus. Stable levels of GPRC5B and RARalpha mRNAs were detected in the placenta and yolk sac. In the placenta, GPRC5B was present in maternal and fetal vascular endothelial cells, stromal cells, fibroblast-like cells and glycogen cells. A strong reaction to RARalpha was detected in maternal and fetal vascular endothelial cells and stromal cells. The levels of GPRC5B and RARalpha proteins in maternal and fetal vascular endothelial cells decreased with gestation. In the yolk sac, GPRC5B and RARalpha proteins were detected in vascular endothelial cells, but their levels did not change during the gestation period. These findings indicate that GPRC5B is involved in RA-dependent morphogenesis/angiogenesis and regulation of extracellular matrix synthesis in the murine placenta and yolk sac.

During the third week of pregnancy, the equine conceptus is enclosed within a capsule, the glycan composition of which changes at around day 16 (ovulation = day 0) when the conceptus becomes immobilized (fixed) in the uterine lumen. Our objective was to characterize the process of fixation by identifying changes in major capsule-associated proteins. Individual equine conceptuses (n = 55) were collected transcervically by uterine lavage between days 13.5 and 26.5. Major proteins extracted from capsules were compared with those in fluids from the uterus and yolk sac by SDS-PAGE. Until day 14, a major capsule-associated protein that migrated at approximately 10 kDa was identified by N-terminal sequencing as equine beta2 microglobulin (beta2M). During fixation, beta2M in the capsule underwent limited proteolysis to an approximately 8 kDa form lacking nine amino acids from the N terminus, and was subsequently degraded. Expression of beta2M mRNA was detected in the yolk-sac wall tissues and endometrium between days 13.5 and 17.5. During this period, beta2M in the capsule was evidently not part of a complex with major histocompatibility complex class 1 heavy alpha chain bands because these were undetectable in the capsule and uterine lavage. Uterocalin (p19) was detected in uterine lavage and capsule throughout fixation, but in yolk-sac fluid only before fixation. These studies indicate that intact beta2M is a major protein associated with the embryonic capsule before fixation, after which it undergoes limited proteolysis to a truncated approximately 8 kDa form that remains in the capsule after the conceptus is immobilized.

A simple method for two-dimensional electrophoresis (2-DE) of rat embryonic protein was described. Rat embryos cultured for 24h from day 10.5 of gestation were used as protein samples. Protein samples were lysed in rehydration buffer and separated by isoelectric focusing with immobilized pH gradient for the first dimension and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the second dimension. The use of the DeStreak Reagent as an antioxidant in the lysis buffer and electrode pads in the isoelectric focusing greatly improved the 2-DE pattern. When an embryo was used as a protein sample, about 800 protein spots were detected by silver staining in a 2-DE gel of the standard format. Eighty-one protein spots were identified by mass spectrometry for a primary 2-DE map. The same method could be applied to yolk sac membranes from the cultured embryos. The present method was considered to be suitable for a concomitant 2-DE analysis in in vitro developmental toxicity studies.

Lutein and zeaxanthin are pigmented oxygenated carotenoids, or xanthophylls, derived from plants and concentrated in the retina of primates and birds. We investigated the transport, distribution and depletion of lutein and zeaxanthin in the plasma and tissues of newly hatched chicks fed xanthophyll-free diets. One-day-old Leghorn chicks were randomly divided into two groups. A control group was fed a diet containing lutein and zeaxanthin (5.2 and 1.7 mg/kg diet, respectively) for 28 days. An experimental group was fed a diet containing no lutein and zeaxanthin for 28 days. Plasma and tissues were analyzed for lutein and zeaxanthin at 28 days (control) and on days 1, 14 and 28 (experimental). At hatching, lutein and zeaxanthin were the predominant carotenoids present in the blood and tissues. As indicated by their similar mass contents, there was complete transfer of these carotenoids from egg yolk to chick. Lutein and zeaxanthin concentrations in the plasma and tissues of chicks fed the xanthophyll-free diet decreased rapidly to almost zero (with a depletion time of seven days [t(1/2)]). In contrast, the retina retained its initial concentrations of lutein and zeaxanthin similar to the control group. meso-Zeaxanthin and cis-zeaxanthin were identified only in the retina. The retina concentrated zeaxanthin over lutein. Lutein and zeaxanthin were selectively retained in the retinas of chicks fed a xanthophyll-free diet. In contrast, the plasma and other tissues lost up to 90% of their original content of xanthophylls. These data emphasize the relative stability of lutein and zeaxanthin in the cone-rich retina where they are present as esters in oil droplets. The tissue depletion suggests the need for a regular dietary intake of lutein and zeaxanthin because of rapid depletion in the body. It is clear that these xanthophylls may have an essential role in the cone-rich retina of the chick as evidenced by their selective retention.

SOX13 is a member of the SOX family of transcription factors. SOX proteins play essential roles in development, and some are associated with human genetic diseases. SOX13 maps to a multi-disease locus on chromosome 1q31-32, yet its function is unknown. Here we describe the temporal and spatial expression of SOX13 protein during mouse organogenesis. SOX13 is expressed in the three embryonic cell lineages, suggesting that it may direct various developmental processes. SOX13 is expressed in the developing central nervous system including the neural tube and the developing brain. Expression is also detected in the condensing mesenchyme and cartilage progenitor cells during endochondral bone formation in the limb as well as the somite sclerotome and its derivatives. SOX13 is also detected in the developing kidney, pancreas, and liver as well as in the visceral mesoderm of the extra-embryonic yolk sac and spongiotrophoblast layer of the placenta.

The roles of extra-embryonic tissues in early vertebrate body patterning have been extensively studied, yet we know little about their function during later developmental events. Here, we analyze the function of the zebrafish extra-embryonic yolk syncytial layer (YSL) specific transcription factor, Mtx1, and find that it plays an essential role in myocardial migration. Downregulating the function of Mtx1 in the YSL leads to cardia bifida, a phenotype in which the myocardial cells fail to migrate to the midline. Mtx1 in the extra-embryonic YSL appears to regulate the embryonic expression of fibronectin, a gene previously implicated in myocardial migration. We further show dosage-sensitive genetic interactions between mtx1 and fibronectin. Based on these data, we propose that the extra-embryonic YSL regulates myocardial migration, at least in part by influencing fibronectin expression and subsequent assembly of the extracellular matrix in embryonic tissues.

We expressed zebrafish p53 protein fused to GFP by a neuron-specific HuC promoter in zebrafish embryos. Instead of displaying neuronal expression patterns, p53-GFP was targeted to zebrafish YSL nuclei. This YSL targeting is p53 sequence-specific because GFP fusion proteins of p63 and p73 displayed neuronal-specific patterns. To dissect the underlying mechanisms, various constructs encoding a series of p53 mutant proteins under the control of different promoters were generated. Our results showed that expression of p53, in early zebrafish embryo, is preferentially targeted to the nuclei of YSL, which is mediated by importin. Similarly, this targeting is abrogated when p53 nuclear localization signal is disrupted. In addition, the transcriptional activity of p53 is required for this targeting. We further showed that fusion of pro-apoptotic BAD protein to p53-GFP led to apoptosis of YSL cells, and subsequent imperfect microtubule formation and abnormal blastomere movements.

Objective: To review the literature to produce a catalog of solutes measured in paired extraembryonic celomic fluid and maternal serum samples., Study Design: We conducted a MEDLINE and PUBMED literature search for January 1990 through March 2004 using the keywords coelomic fluid, celomic fluid, coelocentesis, celocentesis and maternal serum to select articles that met the objective criteria. The following information was extracted from these publications: gestational age at sampling, number of samples, mean concentration of the solute measured in maternal serum and extra-embryonic celomic fluid, authors and year of publication. The catalog is presented in 3 tables based on the gradients found between compartments., Results: We found 73 publications and selected 42 in which paired celomic fluid and maternal serum samples had been obtained. Ninety solutes were grouped into 3 tables using a 30% cutoff difference in favor of either of the 2 compartments., Conclusion: A catalog of solutes measured in human extraembryonic celomic fluid between the 5th and 14th weeks of pregnancy was generated. This catalog can provide guidance for future manipulations of extracelomic fluid for therapeutic purposes.

Insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) are paracrine regulators of tissue growth and development, and are expressed at the sites of biological action. To study the role of the IGFs and IGFBPs in mouse placental development, we determined the temporal and spatial expression patterns of the mRNAs at embryonic days 10.5 to 18.5 by in situ hybridization. IGF-II mRNA was expressed strongly in mesoderm and fetal blood vessels of early placenta and in labyrinthine trophoblast of later placenta. In the junctional zone, IGF-II mRNA was expressed first in spongiotrophoblasts, later strongly in glycogen cells and variably in giant cells. IGFBP-2 mRNA was expressed weakly in spongiotrophoblasts and glycogen cells. IGFBP-2, -5 and -6 mRNAs were detected in the stroma of the metrial gland. Myometrium expressed IGFBP-2 mRNA strongly, IGFBP-6 mRNA moderately and IGFBP-5 mRNA weakly. The endothelium of maternal blood vessels in decidua expressed IGFBP-3 and -5 mRNAs, and some deeper vessels expressed IGFBP-4 mRNA. In the yolk sac, IGF-II mRNA was expressed in endoderm and mesoderm, whereas IGFBP-1, -2 and -4 mRNAs were expressed only in endoderm, and IGFBP-4 mRNA in mesoderm. Strong expression of IGF-II mRNA in glycogen cells suggests a role in the autocrine/paracrine regulation of invasion. Similar to rat and guinea pig, but in contrast to man and primates, IGFBP mRNAs, except IGFBP-4, were not expressed in mouse decidua. However, IGFBP-3, -4 and -5 mRNAs were expressed in endothelium of maternal blood vessels, and IGFBP-2 and -6 mRNAs in myometrium, where IGFBPs may play a critical role in regulating trophoblast invasion. These findings suggest possible biological roles of the peptides at the feto-maternal interface.

Polybrominated diphenyl ethers (PBDEs) used to prevent fire are found in several products, such as textiles, electronics, and building materials. They are lipophilic and persistent substances, and their toxicological endpoints resemble those of polychlorinated bipenyls (PCBs). One of the most abundant congeners of PBDEs in wildlife is the 2,2',4,4',5-pentabromodiphenyl ether (PBDE-99). To study the effect of PBDE-99 on vitamin status in birds and evaluate the possible application of vitamins as biomarkers for use in monitoring of wildlife, eggs of domestic duck (Anas platyrhynchos) were exposed by yolk sac injection to environmental realistic doses of this congener (0.1, 1, or 10 ng/g ww). Levels of retinol (vitamin A), retinyl palmitate, and a-tocopherol (vitamin E) were measured in the liver of the newly hatched chicks, and levels of retinol and tocopherol were also measured in plasma. Liver tocopherol levels correlated negatively to the exposure to PBDE-99. This is an indication that exposure to PBDE-99 reduces levels of tocopherol in liver. Thus, tocopherol seems to be a potential useful biomarker for exposure to PBDEs in bird species.

The study was to investigate the expression of VEGFA, VEGFC, angiopoietin-1, angiopoietin-2 and their receptors on yolk sac blood island, AGM region during gestation of 3th-12th weeks of human embryo. Human embryo contingently aborted at 3 - 12 weeks of gestation were collected with signed agreements of the pregnant women suffered from accidental abortions. The specimens were fixed by 4% paraformaldehyde and embedded by paraffin. 5 micro m serial sections were made. HE and immunohistochemistry method (SABC) and light-microscope were employed. The results showed that VEGFA and its receptors flt1/flk-1, VEGFC and its receptor flt-4, angiopoietin-2 and its receptor tie-2 proteins were expressed strongly and angiopoietin-1 was weakly expressed by hematopoietic cells and vascular endothelial cells of blood island at 21 and 25 days of gestation. In the 4th week of gestation, immuno-positive reaction of these factors and their receptors appeared in the aorta and mesonephros deposited in larger, rounded and nucleated cells which represented hematopoietic cells. Up to 7th week, positive hematopoietic cells in the regions were much abundant. The number of positive cells decreased at 8th week. Up to 12th week, almost all blood cells were immuno-negative. VEGFA, flt-1, flt-4, angiopoietin-1, angiopoietin-2 and Tie-2 protein were expressed mainly by gonad at 6 - 8 weeks, but it did not express VEGFC and flk-1. The immuno-reaction of the factors and their receptors could not detected in vascular endothelial cells during 3-12th weeks of gestation. It is concluded that hematopoietic cells and endothelial cells in blood island of yolk sac, mesonephros and dorsal aorta co-expressed some factors and their receptors in relation to vasculogenesis and hematopoiesis. Intraembryonic hematopoiesis began in the 4th week of gestation.

Background: Folate is essential for cellular proliferation and tissue regeneration. As mammalian cells cannot synthesize folates de novo, tightly regulated cellular uptake processes have evolved to sustain sufficient levels of intracellular tetrahydrofolate cofactors to support biosynthesis of purines, pyrimidines, and some amino acids (serine, methionine). Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking. We utilized in situ hybridization and immunolocalization to determine the developmental distribution of RFC message and protein, respectively., Results: In the mouse, RFC transcripts and protein are expressed in the E10.0 placenta and yolk sac. In the E9.0 to E11.5 mouse embryo RFC is widely detectable, with intense signal localized to cell populations in the neural tube, craniofacial region, limb buds and heart. During early development, RFC is expressed throughout the eye, but by E12.5, RFC protein becomes localized to the retinal pigment epithelium (RPE)., Conclusions: Clinical studies show a statistical decrease in the number of neural tube defects, craniofacial abnormalities, cardiovascular defects and limb abnormalities detected in offspring of female patients given supplementary folate during pregnancy. The mechanism, however, by which folate supplementation ameliorates the occurrence of developmental defects is unclear. The present work demonstrates that RFC is present in placenta and yolk sac and provides the first evidence that it is expressed in the neural tube, craniofacial region, limb buds and heart during organogenesis. These findings suggest that rapidly dividing cells in the developing neural tube, craniofacial region, limb buds and heart may be particularly susceptible to folate deficiency.

The role of thrombopoietin (Tpo) in promoting hematopoiesis has been extensively studied in late fetal, neonatal, and adult mice. However, the effects of Tpo on early yolk sac hematopoiesis have been largely unexplored. We examined whole embryos or the cells isolated from embryo proper and yolk sacs and identified both Tpo and c-mpl (Tpo receptor) mRNA transcripts in tissues as early as embryonic day 6.5 (E6.5). Presomite whole embryos and somite-staged yolk sac and embryo proper cells were plated in methylcellulose cultures and treated with selected hematopoietic growth factors in the presence or absence of Tpo. Tpo alone failed to promote colony-forming unit (CFU) formation. However, in the presence of other growth factors, Tpo caused a substantial dose-dependent reduction in primitive and definitive erythroid CFU growth in cultures containing E7.5 and E8.0 whole embryos and E8.25 to 9.5 yolk sac-derived cells. Meanwhile, Tpo treatment resulted in a substantial dose-dependent increase in CFU-mixed lineage (CFU-Mix) and CFU-megakaryocyte (CFU-Meg) formation in cultures containing cells from similar staged tissues. Addition of Tpo to cultures of sorted E9.5 yolk sac c-Kit(+)CD34(+) hematopoietic progenitors also inhibited erythroid CFU growth but augmented CFU-Mix and CFU-Meg activity. Effects of Tpo on CFU growth were blocked in the presence of a monoclonal antibody with Tpo-neutralizing activity but not with control antibody. Thus, under certain growth factor conditions, Tpo directly inhibits early yolk sac erythroid CFU growth but facilitates megakaryocyte and mixed lineage colony formation.

To gain insight into the role of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes and actions of glucocorticoids in the murine placenta and uterus, the expression pattern of the mRNA for 11beta-HSD1 and 11beta-HSD2 and the glucocorticoid receptor (GR) protein were determined from Embryonic Day 12.5 (E12.5, term = E19) to E18.5 by in situ hybridization and immunohistochemistry, respectively. Consistent with its putative role in regulating the transplacental passage of maternal glucocorticoid to the fetus, 11beta-HSD2 mRNA was highly expressed in the labyrinthine zone (the major site of maternal/fetal exchange) at E12.5, and its level decreased dramatically at E16.5, when it became barely detectable. Remarkably, the silencing of 11beta-HSD2 gene expression coincided with the onset of 11beta-HSD1 gene expression in the labyrinth at E16.5 when moderate levels of 11beta-HSD1 mRNA were detected and maintained to E18.5. By contrast, neither 11beta-HSD1 mRNA nor 11beta-HSD2 mRNA were detected in any cell types within the basal zone from E12.5 to E18.5. Moreover, the expression of 11beta-HSD1 and 11beta-HSD2 in the decidua exhibited a high degree of cell specificity in that the mRNA for both 11beta-HSD1 and 11beta-HSD2 was detected in the decidua-stroma but not in the compact decidua. A distinct pattern was also observed within the endometrium where the mRNA for 11beta-HSD1 was expressed in the epithelium, whereas that for 11beta-HSD2 was confined strictly to the stroma. By comparison, the expression of GR in the placenta and uterus was ubiquitous and unremarkable throughout late pregnancy. In conclusion, the present study demonstrates for the first time remarkable spatial and temporal patterns of expression of 11beta-HSD1 and 11beta-HSD2 and GR in the murine placenta and uterus and highlights the intricate control of not only transplacental passage of maternal glucocorticoid to the fetus but also local glucocorticoid action during late pregnancy.

Experiments were conducted to confirm the existence and ontogeny of fatty acid binding protein (FABP) in the intestine and yolk sac membrane of turkey poults (Meleagridis gallopavo) during embryonic and early posthatch development. Intestinal (I-) FABP was measured using an immunoblot procedure incorporating anti-chick liver (L-)FABP antisera. FABP activity in both tissues was also confirmed with a ligand-binding assay incorporating 14C-oleic acid. I-FABP did not cross-react with chick L-FABP antisera until hatch, embryonic day 28 (ED 28), after which there was a 39% increase in I-FABP concentration through the first 3 d posthatch (PD 3). FABP concentration calculated on a total intestinal basis (ng/intestine), however, increased 10-fold through PD 6. Specific activity [disintegrations per minute (dpm)/ mg cytosolic protein] was greatest at hatch and decreased slightly thereafter, whereas specific activity of FABP in the yolk sac membrane peaked between ED 16 and ED 19 and then declined. Total yolk sac activity (dpm/yolk sac membrane), however, plateaued at ED 22 before declining to low levels by PD 3, coincident with the period of maximal lipid transfer out of the yolk.

The effects of aurofusarin in the quail diet on the antioxidant systems of the developing embryo are investigated. Thirty eight 45-day-old Japanese quails (Coturnix japonica) were divided into two groups and were fed on a corn-soya diet or the same diet supplemented with aurofusarin at the level of 26.4 mg/kg feed in the form of Fusarium graminearum culture enriched with aurofusarin. Eggs obtained after 7 weeks of feeding were incubated. Samples of quail tissues were collected at day 17 of embryonic development and from day old hatchlings. Antioxidants and malondialdehyde were analysed by HPLC-based methods. Inclusion of aurofusarin in the maternal diet was associated with decreased concentrations of alpha- and gamma-tocopherols, alpha- and gamma-tocotrienols, retinol, lutein and zeaxanthin in egg yolk. The vitamin E (tocopherols and tocotrienols) concentration in the liver and yolk sac membrane (YSM) of the day 17 embryos and the hatchlings from aurofusarin-fed group was significantly decreased. Alpha-tocopherol concentration was also reduced in kidney, lung, heart, muscle and brain of day-old quails. In the liver of day-old quails, concentrations of lutein, zeaxanthin, retinol, retinyl linoleate, retinyl oleate, retinyl palmitate and retinyl stearate were also reduced. As a result of these diminished antioxidant concentrations, tissue susceptibility to lipid peroxidation was significantly increased. It is suggested that a compromised antioxidant system of the egg yolk and embryonic tissues could predispose quails to increased mortality at late stages of their embryonic development.

The fetus obtains a significant amount of cholesterol from de novo synthesis. Studies have suggested that maternal cholesterol may also contribute to the cholesterol accrued in the fetus. Thus, the present studies were completed to determine whether diet-induced maternal hypercholesterolemia would affect fetal sterol metabolism. To accomplish this, maternal plasma cholesterol concentrations were increased sequentially by feeding hamsters 0.0%, 0.12%, 0.5%, and 2.0% cholesterol. At 11 days into a gestational period of 15.5 days, cholesterol concentrations and sterol synthesis rates were measured in the three fetal tissues: the placenta, yolk sac, and fetus. In the placenta and yolk sac, the cholesterol concentration increased significantly when dams were fed as little as 0.12% cholesterol (P < 0.0167), and sterol synthesis rates decreased in dams fed at least 0.5% or 2% cholesterol, respectively (P < 0.0167). In the fetus, changes in fetal cholesterol concentration and sterol synthesis rates occurred only when dams were fed at least 0.5% cholesterol, which corresponded to a greater than 2-fold increase in maternal plasma cholesterol concentrations. When the cholesterol concentration in the fetal tissues in each animal was plotted as a function of maternal plasma cholesterol concentration, a linear relationship was found (P < 0.001). These studies demonstrate that sterol homeostasis in fetal tissues, including the fetus, is affected by maternal plasma cholesterol concentration in a gradient fashion and that sterol metabolism in the fetus is dependent on sterol homeostasis in the yolk sac and/or placenta.

Alpha-fetoprotein (AFP) is the major serum protein during development. AFP is one of the earliest proteins to be synthesised by the embryonic liver. The synthesis of AFP decreases dramatically after birth and only trace amounts are expressed in the adult liver. The tissue distribution of AFP in early human embryogenesis has not been defined. We have studied the expression pattern of AFP mRNA in human and mouse embryos by in situ hybridisation. In humans, AFP is expressed in the hepatic diverticulum at 26 d postovulation as it differentiates from the foregut endoderm (i.e. in the most primitive hepatocytes). It is also expressed in the endoderm of the gastrointestinal tract and in the yolk sac at this age. AFP is subsequently expressed in the mesonephros and transiently in the developing pancreas. In the mouse, no expression of AFP was observed in the mesonephros but other sites of expression were similar. Thus AFP has a distinct temporospatial expression pattern during the embryonic period and this differs between human and mouse species. It is interesting that AFP is expressed by tumours such as primitive gastrointestinal, renal cell and pancreatic tumours as well as those of hepatocyte origin. This distribution reflects the sites of AFP expression during development.

1. The fatty acid profile of egg yolk and vitamin E and carotenoid accumulation in the egg yolk and embryonic tissues were investigated in relation to the maternal diet. 2. Two hundred fertile eggs (Ross 308 Broiler Breeder), obtained from hens fed on a maize-based (M-diet) or a wheat-based diet (W-diet), were incubated using standard conditions. 3. The egg yolk and embryo tissues (residual yolk, yolk sac membrane, liver, kidney, lung, muscles, adipose tissue and plasma) were collected on d 18 of incubation and on d 21 (newly-hatched chicks) and analysed for fatty acids, vitamin E and carotenoids. 4. The diets did not differ in terms of fatty acid or alpha-tocopherol concentrations. The concentration of carotenoids in the M-diet was 11.8 mg/kg and in the W-diet was 5.6 mg/kg with lutein and zeaxanthin being major carotenoids. 5. Eggs from the M-group contained higher (P<0.01) concentrations of beta+gamma-tocopherols, total carotenoids, lutein and zeaxanthin. Chickens hatched from those eggs were characterised by the increased concentrations of total carotenoids and zeaxanthin in all the tissues studied. The concentration of beta+gamma-tocopherol was enhanced only in the liver and yolk sac membrane. 6. It is concluded that the maternal diet plays an important role in antioxidant systems formation during chick embryonic development; the M-diet can increase the antioxidant potential of the egg yolk and embryonic tissues compared to the antioxidant potential provided by parent birds fed the W-diet.

Competitive exclusion of intestinal pathogens by administration of beneficial and defined cultures of normal intestinal microflora is a safe and effective means of reducing the incidence and severity of chick infections with Salmonella and other intestinal pathogens. It is important that competitive exclusion cultures not carry genetic material (e.g., plasmids), which could transfer antibiotic resistance to other microflora, including pathogens. As such, safe and effective competitive exclusion cultures must be sensitive to commonly used antimicrobial agents. By necessity, intentional or accidental exposure of these beneficial microflora to antibiotics will reduce or eliminate the protection provided by competitive exclusion culture establishment. As antibiotic residues can be present from embryonic, hatchling, or maternal administration, a rapid and sensitive assay for the nonspecific detection of residues, which could interfere with competitive exclusion culture establishment, is needed. This study was conducted to develop a rapid and inexpensive bioassay to detect multiple antimicrobial residues in egg yolk and neonatal yolk sacs. Aerobic bacterial strains with known sensitivity to several antibiotics used by the poultry industry were selected and individually compared for sensitivity to enrofloxacin, gentamicin, tetracycline, ceftiofur, and tylosin concentrations in egg yolks. This assay was found to be relatively sensitive for the detection of these antimicrobials, and detection of residues was associated with reduced competitive exclusion culture (PREEMPT) establishment in one experiment. Importantly, this assay can be implemented with minimal training or equipment under commercial hatchery practices and could be used to determine embryo groups, in advance of hatch, that are not suitable candidates for competitive exclusion treatment in the hatchery.

Ca(2+), which enters cardiac myocytes through voltage-dependent Ca(2+) channels during excitation, is extruded from myocytes primarily by the Na(+)/Ca(2+) exchanger (NCX1) during relaxation. The increase in intracellular Ca(2+) concentration in myocytes by digitalis treatment and after ischemia/reperfusion is also thought to result from the reverse mode of the Na(+)/Ca(2+) exchange mechanism. However, the precise roles of the NCX1 are still unclear because of the lack of its specific inhibitors. We generated Ncx1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous Ncx1-deficient mice died between embryonic days 9 and 10. Their hearts did not beat, and cardiac myocytes showed apoptosis. No forward mode or reverse mode of the Na(+)/Ca(2+) exchange activity was detected in null mutant hearts. The Na(+)-dependent Ca(2+) exchange activity as well as protein content of NCX1 were decreased by approximately 50% in the heart, kidney, aorta, and smooth muscle cells of the heterozygous mice, and tension development of the aortic ring in Na(+)-free solution was markedly impaired in heterozygous mice. These findings suggest that NCX1 is required for heartbeats and survival of cardiac myocytes in embryos and plays critical roles in Na(+)-dependent Ca(2+) handling in the heart and aorta.