Wyniki wyszukiwania - Biblioteka Nauki

1

Maturation and activation of porcine oocytes in the aspect of fertilization and somatic cell cloning ? biochemical, molecular and biophysical determinants

100%

Samiec M.

Biotechnologia

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2009

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nr 3

59-84

EN

The commonly used source of nuclear recipient cells in the somatic cell cloning of pigs are in vivo-matured (ovulated) or in vitro ? matured oocytes, reversibly blocked at the second metaphase (MII) stage. One of the most important factors that significantly affect the developmental competences of porcine cloned embryos is the artificial activation of oocytes reconstructed with somatic cell nuclei. The ability of an artificial stimulus to activate MII-stage oocytes and to initiate embryo development is essential for successfull cloning by somatic cell nuclear transfer. This ability is especially important for species such as the pig where relatively little is known about early embryonic development and where in vitro handling procedures have not been optimized. An optimal time frame to activate gilt or sow oocytes may depend on both the time required for completion of nuclear-cytoplasmic maturation and the time by which aging process of mature oocytes starts. Cytoplasmic maturation is likely to include changes in the properties, size, and density of cytoplasmic Ca2+ release channels necessary for the oocyte to elicit an increase in intracellular Ca2+ concentration in response to the activating stimuli and subsequent development. Activation of oocytes, which has been induced either during fertilization or by artificial agents during the cloning procedure, evokes the cytosolic calcium concentration ([Ca2+]c) oscilations or single [Ca2+]c transients. Despite the uncertainty of how the initial rises in [Ca2+]c are prompted, it is widely accepted that physiological or artificial activation stimulates the phosphoinositide pathway, with the generation of myo-inositol-1,4,5-trisphosphate (InsP3) by the enzymatic action of phospholipase C (PLC), and the subsequent release of calcium cations from endoplasmic reticulum. Further investigation in nto the role of PLC isoforms as the triggers of [Ca2+]c increases has led to the recent identification of a sperm-specific PLC (PLC-ksi) as the putative sperm-derived oocyte activating factor. It is known that InsP3-mediated calcium signaling pathway is responsible for downregulation of maturation-promoting factor (MPF), which contributes to such events during oocyte activation as resumption and termination of meiosis, extrusion of the second polar body, pronuclear formation, transition from meiotic to mitotic control of cell cycle and initiation of embryonic cleavage.

2

The Bcl-2 family proteins and calcium signaling in mammalian embryos generated by somatic cell cloning

100%

Samiec M.

Biotechnologia

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2010

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nr 1

66-81

EN

The efficiency of somatic cell nuclear transfer (SCNT) technology in mammalian species remains unsatisfactory. One of the main causes of low developmental capability of pre- and peri-implanted somatic cell cloned embryos is the high occurrence of apoptotic cell death, which is prompted by incorrect calcium signaling. The latter is accompanied by upregulation of the members from the Bcl-2 protein family in the blastomeres of SCNT embryos derived from the reconstructed oocytes exposed to artificial activating factors that induce the phenomenon of Ca2+ ion excitotoxicity. Overexpression of antiapoptotic proteins from the Bcl-2 family plays a fundamental role in suppression of different pathways involving intracellular transduction of programmed cell death signal in the somatic cell cloned embryos. Enhancement of Bcl-2 synthesis in the cytoplasm as well as on the outer/cytoplasmic surface of cisterns and tubules of granular endoplasmic reticulum (ERg) and thereby increase in its concentration and activity in the membranes of ER and mitochondria prevents the redistribution of free calcium cations from ER to mitochondria. The purpose of this article is to provide an overview of the current knowledge on molecular aspects of controlling calcium intracellular homeostasis in mammalian SCNT embryos, in which apoptotic cell death was stimulated by an improper activation of reconstituted oocytes.

3

Calcium signal transduction in reconstructed oocytes and somatic cell nuclear transfer embryos of mammals in the conditions of artificial activation

100%

Samiec M.

Biotechnologia

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2010

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nr 1

46-65

EN

One of the most important factors that affect the developmental competences of mammalian somatic cell nuclear transfer (SCNT) embryos is artificial activation of reconstructed oocytes (clonal cybrids). However, calcium signal transduction in a cytosol of such oocytes that has been initiated incorrectly by physical or chemical activating factors (electric pulses or specific ionophore antibiotics) can stimulate not only the development of cloned embryos. It can also induce apoptotic cell death following considerable elevation in intracellular calcium concentration and thereby excitotoxicity of Ca2+ ions. Therefore, the basic objective of this paper is to present the current knowledge on the mechanisms regulating biochemical and biophysical proapoptotic changes within SCNT embryos via the process of excitotoxic calcium signal transmission resulting from an improper artificial activation of clonal cybrid.

4

Artificial activation of porcine oocytes in somatic cell cloning procedure - biotechnological, genetic and epigenetic aspects

100%

Samiec M.

Biotechnologia

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2009

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nr 3

85-110

EN

In the somatic cell cloning of pigs, nuclear transfer-derived oocytes are artificially stimulated with the use one of the three experimental protocols: 1) electrical, chemical or physicochemical delayed activation (i.e., post-activation); 2) simultaneous fusion and electrical activation (SF-EA) or simultaneous electrofusion and physicochemical activation, as well as 3) chemical sequential (combined) electrical and chemical activation. In the first activation protocol, somatic cell nuclei at G0/G1 or G2/M stages are introduced into enucleated Metaphase II oocytes (ooplasts), which are activated 30 minutes to several hours after nuclear transfer. In the second activation protocol, somatic cell nuclei at G1 or G0 stage are introduced into non-activated Metaphase II ooplasts and simultaneously obtained clonal nuclear-cytoplasmic hybrids are activated. In turn, the third activation protocol includes the SF-EA followed by an additional treatment of the reconstituted oocytes with chemical factors, which is initiated after a 1.5-2-h delay. The concentration of calcium cations in the fusion/activation medium affects not only the transition from meiotic to mitotic control of cell cycle of clonal cybrids, but also the degree of ploidy of reconstructed zygotes as a result of both emission of second polar body and formation of pseudopronucleus/pseudopronuclei. The artificial stimulation of reconstituted oocytes also determines the processes of architectural remodeling and epigenetic reprogramming of donor cell nuclei in nuclear-transferred embryos. Moreover, the transcriptional and translational activity of genes (eg Oct-3/Oct-4) that are crucial for preimplantation development of porcine cloned embryos is dependent on physicochemical parameters of calcium oscillations induced by activation of clonal nuclear-cytoplasmic hybrids.

5

The use of new strategies in the cloning studies on somatic cell cloning in pigs

63%

Skrzyszowska M. , Samiec M.

Biotechnologia

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2006

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nr 1

68-81

EN

Somatic cell nuclear transfer (SCNT) technique in pigs remains relatively low (2% to 5% of produced piglets), that is why further efforts have to be made to optimize both a multi-step cloning procedure and to improve a structuro-functional quality of recipient oocytes and nuclear donor cells. Pre- and postimplantation developmental potential of porcine SCNT-derived embryos depends to a high degree on not only coordination of mitotic cycle stage with phenotype of nuclear donor cell, but also proper combination of the methods of maternal chromosome elimination (enucleation), oocyte reconstruction techniques, the systems of artificial activation of generated nuclear-cytoplasmic hybrids (clonal cybrids) and in vitro culture of reconstructed embryos. Generally, it can result in increasing the competences of both somatic nuclear and mitochondrial genome for epigenetic remodeling/reprogramming in developing cloned embryos.

6

Development of the studies on somatic cell cloning in goats

63%

Skrzyszowska M. , Samiec M.

Biotechnologia

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2006

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nr 1

133-150

EN

Domestic goat as a species with a relatively great biodiversity of dairy breeds, which possess high genetic merit and yield of milk production, can be a valuable tool for embryo gene engineering. This involves the generation of transgenic specients, providing with xenogeneic (human) recombinant proteins (i.e. biopharmaceuticals), not only by the standard zygote intrapronuclear microinjection of gene constructs, but above all with the use of somatic cell cloning technology.

7

The role of domestic pig in somatic cell cloning and transgenesis

63%

Skrzyszowska M. , Samiec M.

Biotechnologia

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2006

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nr 1

53-67

EN

A stimulus for development of the studies on pig somatic cell cloning, especially in recent years, was above all the possibility of its practical application for production of transgenic piglets using in vitro transfected nuclear donor cells and multiplication of genetically-engineered sows and boars generated so far, on the grounds of important implications for biomedicine, pharmacy and agriculture. However, effective pig somatic cell nuclear transfer, avoiding the sexual reproduction pathway, creates a possibility of providing numerous monogenetic and monosexual offspring derived not only from genetically-transformed individuals, but also from adult (postpubertal) animals of high genetic merit. Generation of cloned transgenic pigs for biomedical purposes to obtain recombinant xenogeneic proteins or organs suitable in xenotransplantology, or to create cell (gene) therapy foundations for a number of serious monogenic diseases that induce heritable (congenital) developmental anomalies, is perceived as a service to humanity.