Cell population growth in chick blastoderms cultured in vitro

Primitive streak stage chick blastoderms were cultured in vitro up to 30 hr by New's technique. Chick blastoderms reaching stages 4 to 12 in vitro cultures and in ovo were harvested and homogenized to release cell nuclei. Fluorescent ethidium bromide-stained nuclei in homogenates were counted in Neubauer's chamber and the size of total blastoderm cell population was determined. Linear regression analysis revealed that both in ovo and in vitro chick blastoderm cell population grows in a biphasic manner with comparable cell population doubling times and the morphogenesis is not affected in vitro during the culture period.     

Erythropoietin-like factor production by young chick blastoderms.

When grown in liquid culture, chick blastoderm cells release an erythropoietic stimulating factor (ESF) whose properties in vitro and in vivo are very similar to those of erythropoietin found in the serum of anemic adult chick. The role of this factor in the induction of the first blood islands of the chick embryo is discussed.     

Erythrocytic transplantable stem cells in young chick blastoderms.

Young chick blastoderms contain erythropoietic stem cells able to produce normocytic clones when grafted to irradiated chickens. These stem cells are present as early as the 36th hour of incubation, long before the occurrence of the first normoblasts. Later, they exhibit exponential growth.     

The binding of cations to the surfaces of cells from early chick blastoderms

Cells of the early chick blastoderms are either preparing for or undergoing regulated morphogenetic movements which culminate in the formation of a three-layered embryo. Information on the changes in the physical-chemical properties of cell surfaces may help in the understanding of this process. The binding of magnesium, manganese, strontium, barium and lanthanum to surfaces of early embryonic cells was estimated by the changes induced by these cations in the cells' electrophoretic mobilities (EPM). Cells show a positive EPM at concentrations of MgCl2 and MnCl2 at 3 X 10(-2) M while SrCl2 and BaCl2 were not able to reverse the cells' charge at concentrations up to 6 X 10(-2) M. CaCl3 reversed the cells' EPM at concentrations as low as 5 X 10(-3) M. Our results suggest that the surfaces of early embryonic cells have a high affinity for Mg and Mn. This is indicated by a reversal of polarity which cannot be detected in cells of differentiating or adult tissues at the cation concentrations used in these experiments.     

Thymidine incorporation by cultured chick pineal glands is not subject to adrenergic regulation

Norepinephrine is known to play a role in regulating the circadian rhythms of serotonin N-acetyltransferase activity and melatonin formation in the chick pineal gland. We have recently demonstrated that the cultured chick pineal exhibits a circadian rhythm in the incorporation of thymidine. In this study we show that this latter rhythm is not subject to adrenergic control.     

Immunofluorescence localization of plasma protein synthesis in cultured chick hepatocytes.

Fibrinogen, albumin and the major apoprotein of high density lipoprotein (apoprotein A) were localized in a primary embryonic chick liver cell culture by indirect immunofluorescence staining. Changes in the pattern of plasma protein synthesis under a variety of conditions, as measured by the accumulation of secreted plasma proteins in the culture medium, could be studied at the cellular level because relative fluorescence intensities were shown to reflect synthetic rates. In all cases studied, the immunofluorescence of the hepatic parenchymal cells was of a similar intensity throughout the monolayers, indicating that the cells in culture constitute a homogeneous population with respect to the synthesis of these plasma proteins.     

The regulation of the intracellular sodium ion concentration in cultured chick embryo heart cells.

Using digital imaging microscopy with the fluorescent indicator sodium-binding benzofuran isophtalate, we examined the cytosolic Na+ concentration ([Na+]i) in individual chick embryo heart cells. Inhibition of the Na(+)-H+ exchanger using Na(+)-free (Li+ substituted) medium and inhibition of the Na(+)-efflux through the Na(+)-Ca2+ exchanger using Ca(2+)-free medium didn't change the [Na+]i. The opening of voltage-dependent Na+ channels with veratridine (150 micrograms/ml) and inhibition of the Na(+)-K(+)-Cl(-)-cotransporter with bumetanide (10 microM) led to an increase in [Na+]i by 107% and 86%, respectively, suggesting that the Na+ channels and the Na(+)-K(+)-Cl- cotransporter predominantly regulate the [Na+]i in cultured chick embryo heart cells.     

Na/H exchange in cultured chick heart cells. pHi regulation

The purpose of this study was to establish the existence of Na/H exchange in cardiac muscle and to evaluate the contribution of Na/H exchange to pHi regulation. The kinetics of pHi changes in cultured chick heart cells were monitored microfluorometrically with 6-carboxyfluorescein and correlated with Nai content changes analyzed by atomic absorption spectrophotometry; transmembrane H+ movements were evaluated under pH stat conditions. After induction of an intracellular acid load by pretreatment with NH4Cl, a regulatory cytoplasmic alkalinization occurred with a t1/2 of 2.9 min. pHi regulation required external Na+ and was concomitant with transmembrane H+ extrusion as well as a rapid rise in Nai content in an Na/H ratio of 1:1. Microelectrode recordings of membrane potential demonstrated directly the electroneutral character of pHi regulation. Acid-induced net Na+ uptake could be either stimulated by further decreasing pHi or inhibited by decreasing pHo; Na+ uptake was unaffected by tetrodotoxin (10 micrograms/ml), quinidine (10(-3) M), DIDS (10(-4) M), Clo-free solution, or HCO3-free solution. Amiloride (10(-3) M) maximally inhibited both pHi regulation and Na+ uptake; the ID50 for amiloride inhibition of Na+ uptake was 3 microM. Nao-dependent H+ extrusion showed half-maximal activation at 15 mM Nao; Li+, but not K+ or choline+, could substitute for Na+ to support H+ extrusion. Cao-free solution also stimulated acid-induced Na+ uptake. We conclude that pHi regulation following an acid load in cardiac muscle cells is by an amiloride-sensitive, electroneutral Na/H exchange. Stimulation of Na/H exchange up to 54 pmol/cm2 X s indicates the rapidity of this exchange across cardiac cell membranes. Na/H exchange may also participate in steady state maintenance of pHi.     

Depression by hyaluronic acid of glycosaminoglycan synthesis by cultured chick embryo chondrocytes

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ${}^{14}\text{C}{}^3\text{H}$ ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ${}^{14}\text{C}{}^3\text{H}$ incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.     

Steroids and related products. LIV. The synthesis of 11-oxa steroids. VI. The synthesis of 11-oxatestosterone

The synthesis of 11-oxatestosterone from 11-oxa-5 alpha-androstane-3,17-dione, which is available from hecogenin, is described. The product shows, in comparison with the natural hormone, diminished androgenic and anabolic activities.     

The morphogenetic action of follicle-stimulating hormone on post-nodal fragments of early chick blastoderms

Summary The morphogenetic action of ovine follicle-stimulating hormone has been studied. The hormone is incorporated in known concentrations in agar-albumen-compton base and post-nodal fragments of primitivestreak stage chick blastoderms 0.7 mm behind theHensen's node are cultured on the base for 35–45 hours. The hormone is found to induce the formation of neural tissue, notochord-like tissue and mesodermal aggregations resembling somites.     

Steroids and related products. LIII. The synthesis of 11-oxa steroids. V. The synthesis of 17-ethynyl-11-oxatestosterone

The synthesis of 17-ethynyl-11-oxatestosterone, both from 11-oxa-5 alpha-pregnane-3,20-dione and, via a 3,17-dioxygenated 9-oxo 9,12-seco 11-nor 5 alpha-androstane-12-oic ester, from 3 beta-acetoxy-17-hydroxy-5 alpha-pregnan-12-one--two products available from hecogenin--is reported. The new hormone analogue shows significant progestational activity in the Clauberg test and relatively weak activity in a post-coital antifertility assay.