Direct yolk sac volume manipulation of zebrafish embryos and the relationship between offspring size and yolk sac volume

Through direct manipulation of yolk sac volume ( V _y), in the zebrafish Danio rerio , V _y and offspring size was positively correlated and the relationship was independent of geographical location, parental size or age and, most importantly, parental genetic factors. Larval survivorship, under a non-feeding regime and up until hatch, was not significantly affected by the manipulation. Decreased V _y significantly decreased maximum standard length ( L _S), maximum body surface area ( A _B), time to yolk sac absorption, L _S and A _B at yolk sac absorption, and the L _S and A _B at starvation. The methodology of V _y adjustment will be useful for the studies of the interaction between early life-history traits and offspring size, egg quality variables and early vertebrate development.     

Synthesis of secretory proteins in developing mouse yolk sac

Synthesis of secretory proteins in the developing mouse visceral yolk sac was studied. Newly synthesized proteins were labeled with [³⁵S]methionine and characterized by two-dimensional gel electrophoresis. A large increase in the relative rate of synthesis of a small number of proteins occurred between Days 9.5 and 15.5 of development. These proteins were the predominant proteins synthesized and secreted by the yolk sac throughout this period of gestation. Two of these proteins were identified as α-fetoprotein and transferrin by specific immunoprecipitation. α-Fetoprotein synthesis increased from about 3% of the total protein synthesis at Day 9.5 to about 26% at Day 15.5 after which it declined slightly. The relative rate of transferrin synthesis had a similar developmental pattern, reaching the highest level (5%) at Day 15.5, but declined more rapidly than α-fetoprotein synthesis. Quantitatively, these two proteins represented about 60% of the total secreted protein. Gestational changes in the content of α-fetoprotein messenger RNA were determined by hybridization analysis using α-fetoprotein complementary DNA probe. The percentage of α-fetoprotein messenger RNA in total yolk sac RNA increased about ninefold from Day 9.5 to Day 14.5. This increase correlated well with the increase in the relative rate of α-fetoprotein synthesis during the identical period. This study suggests that after Day 9.5 the yolk sac is completing a differentiation process which is characterized by the preferential expression of a small group of secretory protein genes.     

Mineral metabolism in the developing turkey embryo--II. The role of the yolk sac.

1. Turkey embryos were incubated in ovo or in long-term shell-less culture (ex ovo) for 14, 18, 22 or 26 days, at which time the concentrations of zinc, copper, iron, manganese and calcium in yolk and yolk sac membrane were determined. 2. Yolk manganese and calcium concentrations increased during incubation in ovo while the concentrations of zinc, copper and iron declined. The concentrations of zinc, copper and iron in yolk from ex ovo embryos did not decline. Yolk calcium concentration increased during incubation ex ovo, although to a much lesser extent than that observed in ovo. 3. The concentration of zinc, copper and iron declined in yolk sac tissue during incubation in ovo whereas no decline was observed for yolk sac tissue from ex ovo embryos. Yolk sac calcium and manganese concentrations increased during incubation in ovo and ex ovo, although the increase in calcium concentration for ex ovo yolk sac was much smaller than that observed in ovo. 4. A peak corresponding to metallothionein (MT) which bound both zinc and copper was isolated from yolk sac cytosol on day 14 of incubation in ovo using gel-permeation column chromatography. 5. Further fractionation of the MT peak by anion exchange chromatography revealed three metal-binding peaks designated MT-1, MT-2a and MT-2b. The majority of the zinc was bound to MT-2a and MT-2b whereas most of the copper was bound to a single peak (MT-2b). 6. The concentrations of zinc and copper in yolk sac cytosol reached a maximum on day 14 of incubation in ovo and declined through to day 28 (hatching).(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of teratomas from yolk sac of genetically sterile embryos

Yolk sac-derived teratomas are composed of various well-differentiated tissues. These tissues must be derived from multipotent cells. To exclude a germ cell origin for these teratomas we used $\text{Steel}\text{-}\text{Sl}\text{+}$ females copulated with $\text{Sl}\text{+}$ males. The embryos generated from such mating comprise 25% $\text{Sl}\text{Sl}$ sterile embryos, deficient in primordial germ cells, 25% normal embryos (+/+), and 50% heterozygotes ($\text{Sl}\text{+}$). The results indicate that the genotype of the embryos does not influence the development of teratomas. Displaced yolks sacs belonging to genetically sterile embryos developed into teratomas as frequently as those from heterozygotes and from genetically normal embryos.     

The role of the visceral yolk sac in hyperglycemia-induced embryopathies in mouse embryos in vitro.

The adverse developmental effects of hyperglycemia to rodent embryos have been shown using whole embryo culture. Although, a mechanism by which hyperglycemia-induced effects occur is unknown, recent work has focused on the visceral yolk sac as a potential target tissue. Therefore, we have evaluated the developmental effects of hyperglycemia in early head fold stage mouse embryos in vitro and assessed the histiotrophic function of the visceral yolk sac. As has been previously shown in rodents, hyperglycemia produced neural tube closure defects in a concentration dependent manner at 33, 50, and 67 mM glucose using a 44 h exposure period. However, exposure times between 6 and 12 h were sufficient to alter embryonic development when the glucose concentration was 50 or 67 mM. In contrast, early somite stage embryos (4-6 somite stage) appear to be less sensitive to dysmorphogenesis and a 48 h exposure to 67 mM glucose but not 33 or 50 mM also produced neural tube defects. Hyperglycemia (67 mM) did not alter the uptake of 35S-methionine and 35S-cysteine-labeled hemoglobin (35S-Hb) in the visceral yolk sac (VYS) in early headfold staged embryos. However, the accumulation of 35S in the embryo was reduced by 16-18% at glucose concentrations of 50 or 67 mM during the last 12 h of a 44 h exposure period. No effect on VYS uptake or embryonic accumulation of 35S-labeled products was observed at shorter exposure periods (12-24 and 24-36 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of the yolk sac in craniofacial development of cultured rat embryos

To study the role of the yolk sac and amnion in craniofacial development, the effects of opening the yolk sac and amnion on facial formation of rat embryos were examined in vitro. Rat embryos were cultured for 72 hr from day 11.5 of gestation using an improved rotation apparatus. In experiments, the yolk sac and amnion were opened at the time of explantation (day 11.5) in one group (D11 open) and were opened 24 hr after the beginning of the culture (day 12.5) in another group (D12 open). Cleft lip developed in 100% of cultured embryos when the yolk sac and amnion were opened at day 11.5 (D11 open). In the D12 open group, however, cleft lip occurrence decreased to 3.0%. Protein content, wet weight, and somite number of cultured embryos were not significantly different in the two groups. The results of this study demonstrate that it is beneficial to open the yolk sac and amnion after 24 hr in culture for normal facial formation of rat embryo cultured from day 11.5 of gestation.     

Duck hepatitis B virus requires cholesterol for endosomal escape during virus entry

The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.     

Duck hepatitis B virus infection of Muscovy duck hepatocytes and nature of virus resistance in vivo.

To test the hypothesis that in vivo resistance to hepadnavirus infection was due to resistance of host hepatocytes, we isolated hepatocytes from Muscovy ducklings and chickens, birds that have been shown to be resistant to duck hepatitis B virus (DHBV) infection, and attempted to infect them in vitro with virus from congenitally infected Pekin ducks. Chicken hepatocytes were resistant to infection, but we were able to infect approximately 1% of Muscovy duck hepatocytes in culture. Infection requires prolonged incubation with virus at 37 degrees C. Virus spread occurs in the Muscovy cultures, resulting in 5 to 10% DHBV-infected hepatocytes by 3 weeks after infection. The relatively low rate of accumulation of DHBV DNA in infected Muscovy hepatocyte cultures is most likely due to inefficient spread of virus infection; in the absence of virus spread, the rates of DHBV replication in Pekin and Muscovy hepatocyte cultures are similar. 5-Azacytidine treatment can induce susceptibility to DHBV infection in resistant primary Pekin hepatocytes but appears to have no similar effect in Muscovy cultures. The relatively inefficient infection of Muscovy duck hepatocytes that we have described may account for the absence of a detectable viremia in Muscovy ducklings experimentally infected with DHBV.     

Fluorescent histochemical demonstration of cathepsin B in the rat yolk sac

Summary Fluorescence demonstration of cathepsin B (E.C. 3.4.22.1) activity was performed in the yolk sac of rats near term (18th day of gestation). The enzyme demonstration was performed on freeze-dried and celloidin mounted yolk-sac sections using different substituted -naphthylamide derivatives as substrates and nitrosalicylaldehyde as coupling agent. The discrete reaction products are localized preferentially in the apical part of the visceral yolk-sac epithelium. There is little doubt that cathepsin B is contained here in the well developed lysosomal apparatus of the yolk-sac epithelium.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Fluorescent histochemical demonstration of cathepsin B in the rat yolk sac

Fluorescence demonstration of cathepsin B (E.C. 3.4.22.1) activity was performed in the yolk sac of rats near term (18th day of gestation). The enzyme demonstration was performed on freeze-dried and celloidin mounted yolk-sac sections using different substituted beta-naphthylamide derivatives as substrates and nitrosalicylaldehyde as coupling agent. The discrete reaction products are localized preferentially in the apical part of the visceral yolk-sac epithelium. There is little doubt that cathepsin B is contained here in the well developed lysosomal apparatus of the yolk-sac epithelium.     

Duck hepatitis B virus polymerase acts as a suppressor of core protein translation.

Nucleocapsid assembly in hepadnavirus replication requires selective encapsidation of the pregenomic RNA template and the viral polymerase by the core proteins. It has been shown that an encapsidation signal located at the 5' end of the pregenomic RNA is responsible for its interaction with the polymerase. In the present study, we have shown that a region located at the 3' periphery of the core open reading frame may interact with the viral polymerase in duck hepatitis B virus. By using an in vitro rabbit reticulocyte lysate translation system, we found that interaction of the polymerase with this region resulted in selective suppression of core mRNA translation. Insertion of this putative inhibitory sequence into the CD4 gene also led to a selective inhibition of CD4 mRNA translation in the presence of polymerase. Specific inhibition of core protein synthesis was observed in a chicken hepatoma cell line (LMH) cotransfected with core and polymerase plasmid DNA.     

Duck hepatitis B virus infection of hepatocytes is not dependent on low pH.

The pH dependency for initiation of infection by the hepadnavirus duck hepatitis B virus (DHBV) was investigated in primary duck hepatocytes. First, an infection assay was developed using a radioimmunoblot to measure DHBV e antigen secreted into tissue culture fluid from infected hepatocytes. The quantity of this viral marker was proportional to the duration of inoculation and the amount of DHBV used as inoculum. The role of pH in initiation of DHBV infection was investigated by using this assay, but no dependence on low pH was found. DHBV was able to infect hepatocytes in the presence of NH4Cl and monensin, agents that raise the pH in intracellular vesicles and prevent penetration of viruses dependent on low pH in endosomes. In control experiments, infection by Semliki Forest virus, which is low pH dependent, was inhibited, whereas herpes simplex virus type 1 infection, which is pH independent, occurred. Attempts to trigger DHBV-cell fusion by exposure of DHBV prebound to hepatocytes to mildly acidic pH were unsuccessful. In these experiments, it was also observed that internalization of DHBV occurred only between pH 6.8 and 8.0. Additionally, in the absence of cells, infectivity of DHBV was stable at pH 4.6 to 4.8, which is lower than the pH encountered in endosomes (pH 5 to 6.6). Thus, no evidence for a role for mildly acidic pH in the initiation of DHBV infection was found. Therefore, we propose that the infection route followed by DHBV resembles that of the group of enveloped viruses, including herpesviruses, that fuse with their host cells at neutral pH.     

Incorporation of a phospholipid precursor into the hemochorionic and yolk sac placentas associated rat embryos.

Pregnant Long-Evans rats were subjected to a teratogenic regimen, i.e., were fed a synthetic diet lacking folic acid and containing 9-methylpteroylglutamic acid on the 11th to 14th days of gestation. Experimental and control pregnant rats injected with 10 muCi of [2-14C] ethanolamine on the 14th day were killed 1 or 2 days later. The total radioactivity and radioactivities of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and lysophosphatidylethanolamine (LPE) were determined in chloroform extracts of homogenates and subcellular fractions prepared from hemochorionic and yolk sac placentas and maternal liver. The distribution of radioisotope into PC and PE of control and experimental yolk sac placentas was similar, and paralleled the distribution in maternal liver. However, the distribution of radioisotope into PC and PE of the hemochorionic placentas did not parallel that of the maternal liver, and radiolabeled PC accumulated faster in experimental placentas than in controls. We suggest that the ability of the hemochorionic placenta to synthesize PC from PE was impaired by the teratogenic regimen, and that the organ took up relatively more PC from the maternal plasma. We propose that this teratogen-induced shift from placental lecithin synthesis to selective lecithin uptake underlies the previous finding of an increased accumulation of radio-labeled PC in embryos from pregnant females subjected to this teratogenic regimen (Chepenik and Waite, '73).