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.     

A study of the inductive capacity of post-nodal primitive-streak pieces after treatment with follicle-stimulating hormone

Summary Chick embryos at the primitive-streak stage were treatedin vitro with 0.002 and 0.008% follicle-stimulating hormone (FSH)(NIH-FSH-S1) for 24 hours. Post-nodal primitive-streak pieces 0.8 mm behind the node level were grafted into host chick embryos at the primitive-streak stage to assess the capacity of the grafted pieces to produce inductions.Control grafts from donors maintained under identical conditions were unable to cause induction; all of them were resorbed into the host embryo. The post-nodal pieces treated with FSH acquired capacity to induce neural tissue. The grafts seemed to induce foregut formation also. FSH appeared to have supported differentiation of the grafts into somites and mesenchyme.     

Changes in the electrophoretic mobility of cells from chick blastoderms during early development

A change in cell surface charge density during early avian development is shown with a free-flow electrophoresis apparatus. The blastoderms of freshly laid eggs consist of two electrophoretically distinct cell populations. After the onset of gastrulation a third cell population with an intermediate electrophoretic mobility appears. With increasing time of incubation there is a shift in the proportion of these populations and an increase in the mobility of the fastest fraction.     

The all-or-none rule in morphogenetic action of juvenile hormone on insect epidermal cells

We have investigated ultrastructural changes in the integuments of larval–adult and larval–pupal intermediates produced by exogenous application of juvenile hormone (JH) analogues in Pyrrhocoris apterus (Hemiptera), and Galleria mellonella and Manduca sexta (Lepidoptera). Ultrastructural analysis of the epidermis of these intermediates always revealed the presence of only two types of epidermal cell, which produced morphologically perfect cuticles of the previous and future developmental stages. There were no intermediate cuticles at the level of individual cells. It has been determined that a single epidermal cell constitutes the lowest elementary unit in the perception and realization of the developmental messages conveyed by JH to its target tissues. Further investigations revealed that the responses of individual epidermal cells to JH were strictly autonomous and qualitative, i.e. they were executed according to the ''yes-or-no'' or ''all-or-none'' rule. The neighbouring epidermal cells could realize independently, side-by-side, the quite dissimilar +JH (somatic growth) or -JH (metamorphosis) developmental programmes, although each of them formed biochemically, functionally, and ontogenetically different structures. The qualitative on- and off- signal given by JH for induction of the stationary (+JH) developmental cycle was limited to relatively short, genetically determined, and stage-specific developmental periods of cellular susceptibility to JH. The mosaic mixtures of the heterochronic, larval–pupal or adult epidermal cells, which we found in different proportions on the bodies of the intermediates, revealed two variable, development-related factors: (i) the presence or absence of a minimum effective concentration of JH, and (ii) positive or negative sensitivity of a particular epidermal cell to JH.     

Tracking morphogenetic tissue deformations in the early chick embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to study the physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.     

Chemical synthesis of peptide fragments of the hormone-specific beta-subunit of human follicle-stimulating hormone

In order to determine the specific antigenic determinants of human follicle-stimulating hormone (hFSH), hFSH-beta peptides with amino acid residues 33-49 (V2), 95-118 (V3), 76-118 (V3 + 1/2 C2), 1-33 (V1 + C1), 22-33 (1/2C1), and 95-107 (V3 + 1/4C2) according to the nomenclature of Stewart and Stewart [Stewart, M., & Stewart, F. (1977) J. Mol. Biol. 116, 175] as well as additional peptides with the residues 93-107, 91-107, 89-107, 87-107, and 85-107 were chemically synthesized. The peptides were examined in radioimmunoassay systems of FSH, luteinizing hormone (LH), or human chorionic gonadotropin (hCG). V3 + 1/2C2 and V1 + C1 showed immunological activity, whereas the other peptides did not. Antibodies were raised in rabbits against these peptides and examined for specific binding with hFSH, LH, thyroid-stimulating hormone (TSH), and hCG. V3 + 1/2C2 as well as V1 + C1 produced antisera, which specifically bound hFSH, hLH, and hTSH, indicating that the amino acid sequences contained in hFSH-beta peptides V3 + 1/2C2 and V1 + C1 share common antigenic sites with hLH and hTSH. Antisera were produced in rabbits against hFSH-beta, against reduced and S-aminoethylated hFSH-beta (AE-FSH-beta), and against AE-FSH-beta coupled to hemocyanin. Reduced and S-aminoethylated beta-subunit of FSH-beta coupled with hemocyanin produced antisera in rabbits that specifically bound only hFSH and not hLH, hTSH, or hCG.     

Effect of gonadotropin-releasing hormone antagonists on serum follicle-stimulating hormone and luteinizing hormone under conditions of singular follicle-stimulating hormone secretion

Previous work has indicated that in long-term ovariectomized rats a potent antagonist to gonadotropin-releasing hormone (GnRH) suppressed serum luteinizing hormone (LH) more successfully than follicle-stimulating hormone (FSH). The present studies examined whether the rise in serum FSH which occurs acutely after ovariectomy, or during the proestrous secondary surge, depends on GnRH. In Experiment A, rats were ovariectomized at 0800 h of metestrus and injected with (Ac-dehydro-Pro1, pCl-D-Phe2, D-Trp3,6, NaMeLeu7)-GnRH (Antag-I) at 1200 h of the same day, or 2 or 5 days later. Antag-I blocked the LH response completely, but only partially suppressed serum FSH levels. Experiment B tested a higher dose of a more potent antagonist [( Ac-3-Pro1, pF-D-Phe2, D-Trp3,6]-GnRH; Antag-II) injected at the time of ovariectomy. The analog suppressed serum LH by 79% and FSH by 30%. Experiment C examined the effect of Antag-II on the day of proestrus on the spontaneous secondary surge of FSH, as well as on a secondary FSH surge which can be induced by exogenous LH. Antag-II, given at 1200 h proestrus, blocked ovulation and the LH surge expected at 1830 h, as well as increases in serum FSH which occur at 1830 h and at 0400 h. Exogenous LH triggered a rise in FSH in rats suppressed by Antag-II. In Experiment D proestrous rats were injected with Antag-II at 1200 h and ovariectomized at 1530 h. By 0400 h the antag had suppressed FSH in controls, but in the ovariectomized rats, a vigorous FSH response occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

The asparagine-linked sugar chains of human follicle-stimulating hormone

The asparagine-linked sugar chains of human follicle-stimulating hormone (hFSH) were liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Ninety-five percent of the oligosaccharides were acidic and all were converted to a mixture of neutral oligosaccharides on sialidase treatment. The mixture of neutral oligosaccharides was subjected to sequential immobilized lectin column chromatography on E-PHA-agarose, AAL-Sepharose, and Con A-Sepharose, and six fractions were obtained. The results of sequential exoglycosidase digestion of each oligosaccharide and methylation analysis led us to propose that the asparagine-linked sugar chains of hFSH are a mixture of complex-type bi-, tri-, and tetraantennary sialylated sugar chains with and without a fucose residue linked at the C-6 position of the proximal N-acetylglucosamine. Some of these sugar chains contain bisecting N-acetylglucosamine residue.     

The regulation of haemoglobin synthesis in cultured chick blastoderms by steroids related to 5beta-androstane.

1. After 24h of incubation, the blastoderm may be dissected from the early developing chick embryo and successfully maintained under conditions of organ culture in vitro. 2. Low concentrations of steroids related to 5beta-androstane stimulate the synthesis of foetal haemoglobins, types E and P, in a highly steroid- and tissue-specific manner.