Embryology - Gradient theory

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Gradient theory & Nuclear Transplantation Dr.G.Ponraj Associate professor Vivekananda college Thiruvedakam - West

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Synopsis 1.0 Definition 2.0 Types of Gradient theory 2.1 Single Gradient theory 2.2 Double Gradient theory 3.0 Experimental Evidences 4.0 Factors Affecting G radients 5.0 Nuclear Transplantation 5.1 Types of Transplantation 5.2 Transplantation technique 6.0 Transplant with experimental evidence 7.0 Reference

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Definition: The Gradient is an embryonic differentiation is the result of gradation in the potentialities for development of various parts of the embryo of such nature that successful differentiation of a part inhibits the potentiality for similar change elsewhere in the system . It is a proportional rise and fall of certain factors in the embryo, the interaction of which brings about normal development. The word gradient is derived from Latin word gradi means to stop. The early development of embryos is controlled by one or more factors called gradients. Control the normal development.  

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Types of Gradient Theory: There are two types, namely 1. Single gradient theory or Metabolic axial gradient theory: This theory was proposed by C.M. Child (1940). He showed that development and morphogenesis are controlled by a single factor. The oxidative metabolic is higher at the animal pole and it gradually decreases towards the vegetal pole along the median axis. The existence of this gradient is proved by the reduction of Janus green.

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2. Double gradient theory: By Runnston and Horstadius (1928). States that the sea urchin egg has two factors or principles. They are the Animal gradient and the vegetal gradient. The animal gradient has its centre of activity at the vegetal pole. The animal gradient diminishes towards the vegetal pole and the vegetal gradient diminishes towards the animal pole; they overlap throughout the whole egg. They are mutually antagonistic; yet they interact. The normal development is due to a certain equilibrium between the two gradients and known as double gradient theory.

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Experimental Evidences: The exdence of the double gradient theory and its mechanism can be proved in two ways: 1. Isolation and recombination of blastomeres: Runnstrom and Horstadius carried out a series of experiments in sea urchin embryo to illustrate the existence of gradient system. Experiment: The first two cleavage are meriditional and they produce four equal blastomeres. Result: When the four blastomeres are separated, each blastomeres develops into a normal larva. This is because all the blastomeres receive equally both a.pole & v.pole.

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Fig: Sea urchin, Separation of blastomeres in 2 and 4 - cell stage.

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2. Physico – chemical Experiments: Experiment : The presence of the gradient system in the developing embryos is demonstrated by physic – chemical experimental. Horstadius (1952) carried out fantastic experiments on sea urchin blastula. Sea urchin embryos are stained in Janus green.( diethyl safranin azo dimethyl aniline) The stained embryos are placed in a dish sealed with petroleum jelly. The embryos respire using Janus green as a receptor of H + . Result: The blue colour is reduced to a red dye - dimethyl safranin. Colorless substance – leucosafranin. Change the colour is first seen vegetal pole – Red colour spreads out gradually and reaches the equator. At this time, Extreme change blue colour into red the animal pole and When the blue colour disappears completely.

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Fig: Reduction of Janus green in an animalized sea urchin embryo.

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F actors Affecting Gradients: The chemicals may suppress either the animal gradient or the vegetal gradient. 1. Animalizing Agents: Certain chemicals cause predominant development of animal pole structures. If, before fertilization, the sea urchin egg is exposed to artificial seawater lacking calcium ions but containing sodium thiocynate (NaSCN) the embryo is animalized. Some metals like Zinc, Mercury, etc. Proteolytic enzymes like trypsin, Chymotrypsin, etc. Some anionic detergents, etc. Mode of action: In this agents either attack the proteins or form compounds with proteins. Block the active site of proteins, inhibiting them from interacting with other cell components. ( Lallier , in 1952,57).

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2. Vegetalizing Agents: Cause predominant development of vegetal pole structures. Seawater containing some lithium salts, Sodium azide, Dinitrophenol, etc. Mode of action: Sodium azide and dinitrophenol are enzymes poisons. Azide inactivates the cytochrome oxidase system while dinitrophenol disturbs respiration by preventing oxidative phosphorylation. When there is no oxidative phosphorylation. ATP molecules is not produced.( Horstadius , in 1953).

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Nuclear transplantation: Definition: It refers to the isolation of a nucleus or a piece of tissue from one embryo and grafting in to another embryo or to another place in the same embryo. The individual donating nucleus or tissue called donor, receiving it is called recipient or host. In this experiments are carried out in a variety of animals such as Amoeba, Rana, Xenopus, Drosophila, Acetabularia, etc. Somatic cell nuclear transfer (SCNT) is a method of cloning of an organism that involves removing the nucleus from a somatic cell and putting it in an egg cell.

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Steps of nuclear transplantation: Nucleus is taken out of organism that is being cloned. Nucleus of an egg cell is removed. The organisms nucleus is placed into the egg cell. The new nucleus get reprogrammed as it were an egg cell. The egg cell is stimulated with a shock. The egg cell starts to divide.

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Limitations: It takes a lot of tires to get a successful clone. The procedure is manual. Some of the wanted DNA is not transferred. Some of the original egg’s DNA is left behind. Result in a close, but not perfect clone. Controversy is a requirement of eggs from a female.

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Types of Transplantation: Autoplastic Transplantation : When a piece of nucleus or tissue is removed and transplanted into another place in the same embryo. Homoplastic Transplantation : If transplantation is done from one individual to another individual of the same species. Heteroplastic Transplantation : Between two species belonging to the same genus. Xenoplastic Transplantation : Between two different genus.

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Transplantation technique: Briggs and king transplanted nucleus of frog embryos into the eggs. 3 types, namely 1. Preparation of Recipient Egg: A ripe egg is removed from a female frog. Pricking with a glass needle. As a result, the egg nucleus moves to the surface of the egg at the animal pole. This nucleus is carefully removed with a micro needle. Now the egg is said to be enucleated. ( Recipient cell). Donor nucleus fig: Nuclear transplantation experiment A B C D E Extra – ovate protrusion Blastula cells Microneedle Egg - nucleus F

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2. Isolation of Donor Nucleus: The embryo from which the nucleus is isolated. The embryo is treated with trypsin in a solution which is free from calcium and magnesium. This treated causes the cells of the embryo to separated from one another. Particular cell is drawn up into the tip of a micropipette. Cell is drawn, the cell surface is broken out. Nucleus surrounded by its own cytoplasm remains inside the pipette.

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3. Injection of Donor Nucleus into the Recipient Egg: Donor nucleus, in the pipette, is injected deep into the enucleated egg. Pipette is drown out, the egg – cytoplasm tends to ooze out in the form of a protrusion called extra – ovate protrusion. Egg starts development immediately. They undergo cleavage, blastulation, gastrulation and are hatched into tadpoles. It develops into the adult frogs. The embryos developed from the transplanted nuclei, transplant embryos.

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Fig: Transplantation technique in Frog embryo and developmental stages of tadpole.

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T ransplantation of Experiments in Amphibians: Gurdon (1968) obtained adults by transplanting nuclei from intestine of the tadpoles of Xenopus. Result: The nuclei of the cleavage and blastula stages are totipotent and equipotent. The totipotency of the nucleus is reduced when the blastula is transformed into gastrula. In Rana, the nuclei taken from embryonic stages later blastula cannot support the normal process of development. But in nucleus of Xenopus any stage support the process. As development proceeds, the totipotentiality of nuclei is limited. The nucleus changes during development, irreversible.

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Fig – 1: Nuclear transplantation experiments in frog tadpoles. Fig – 2: Nuclear transplanted in egg into the Xenopus

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Reference: 1.o Development biology, N. Arumugam, Second Edition 2014, Saras publication, Kanyakumari. P – 273 – 280. 2.0 Development biology, Veer Bala Rastogi, Kedar Nath Ramnath Publication, Meerut. P – 49 &155. 3.0 Embryology, B.I. Balinsky, Fifth Edition, CBS College Publication, 1981, Delhi. P – 181.

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