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The effect of different activation modes on DNA integrity of porcine M II oocytes matured in vitro
14
ETS Abstracts 2008 / Reproductive Toxicology xxx (2008) xxx–xxx
Apoptotic influence of cycloheximide an endocrine disruptor in lymphocytic organs during pregnancy
The effect of different activation modes on DNA integrity of porcine M II oocytes matured in vitro
E. Nikoloussis 1 , S. Massourides 2 , H. Frangou 2 , E.-N. EmmanouilNikoloussi 1
Bozena Novotna 1 , Marketa Sedmikova 2 , Jaroslav Kratochvilova 2 , Frantisek Jilek 2
1 Laboratory of Histology-Embryology and Anthropology, Faculty of Medicine, Aristotle University of Thessaloniki, Greece 2 Laboratory of General Biology, Faculty of Medicine, Aristotle University of Thessaloniki, Greece
1
Pregnancy and nutrition is a complex situation. Nutrients unfortunately come several times extracted from plants which have been recently vaporised with pesticides and fungicides. Cycloheximide [4-{(2R)-2-[(1S,3S,5S)-3,5-dimethyl-2oxocyclohexyl]-2-hydroxyethyl}piperidine-2,6-dione] and Molecular formula C15 H23 NO4 ; is a highly toxic agent and a protein synthesis inhibitor that acts specifically on the 60S subunit of eukaryotic ribosomes. It has been also been discussed as an endocrine disruptor as a common used fungicide. Although toxic, cycloheximide is a widely used fungicide, which must be very carefully applied due to diverse side effects possessing as a pesticide/infecticide drug for plants and fruits. The aim of this study was the Molecular and Transmission Electron Microscope analysis of lymphocytic organs of rats treated with cycloheximide during pregnancy. Cycloheximide (Sigma) was injected at 10 pregnant Wistar rats in dosage level of 3 mg/kg b.w. on 10th and 11th gestational days. Pregnancy on most of the rats resulted in abortions, absorptions and teratomas, while on two pregnant rats that were not sacrificed during pregnancy big breast tumors developed rapidly. Three months after the tumors were first observed, the animals were anesthetized the tumors were removed by a surgical operation and thereafter were sacrificed. Lymphocytic organs, i.e. spleen and lymph nodes were collected and proceeded for molecular and Transmission Electron Microscopical analysis to evaluate the role of the drug in apoptosis on spleen and lymph nodes. Animals’ spleen and lymph nodes as organs and tissues related to the tumors, were removed. For molecular evaluation of caspase-3 activity they were homogenized with a Polytron Kinematika homogenator in sucrose isotonic solution. Following subcellular fractionation according to the method of Nordlie and Lardy, the caspase-3 activity was determined in crude, nuclear, and cytosolic plus ribosomal fraction by a colorimetric assay (kit, Sigma), based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroaniline (pNA) moiety. Caspase-3 activity was calculated in mol pNA released per min per ml. Protein concentration was determined by the method of Bradford, modified by Bearden. Transmission Electron Microscopical analysis was performed under routine procedures for observations at Transmission Electron Microscope (TEM). Ultrastructural observations from spleen and lymph nodes cell parenchyma from pregnant rats showed severe pathological effects and increased apoptotic activity, accumulation of large secondary lysosomes and apoptotic bodies within cells.
Petr 3 , Jana
Institute of Experimental Medicine, CAS, v.v.i., Prague, Czech Republic University of Life Science, Prague 6, Czech Republic 3 Institute of Animal Production, v.v.i., Prague, Czech Republic 2
Introduction: Mammalian embryonic development requires an activating stimulus to overcome the block of oocytes at the stage of metaphase II. However, the physiological nature of this stimulus has not been fully clarified yet. This is one of the reasons why the procedures such as in vitro fertilization or cloning of mammals using the nuclear transfer are still characterized by a relatively low efficiency. Genomic integrity is also a prerequisite of successful development. We decided, therefore (1) to investigate the effect of different activation protocols on DNA of porcine oocytes matured in vitro using a nitric oxide donor (+)–S-nitroso-N-acetylpenicillamine (SNAP) as activating stimulus and (2) to relate the findings with the other parameters of developmental competence. Methods: The oocytes aspirated from ovaries of slaughtered gilts were cultured for 48 h in modified M 199 medium supplemented with gonadotropins. Then, the oocytes were devoid of cumulus cells and subjected to continuous (6 h) or repeated treatment with SNAP (10 min exposure to SNAP alternated with 10 min cultivation in free medium, the cycle was repeated 12 times and terminated by 2 h cultivation in free medium). Standard activation with calcium ions and culture without any treatment was used as positive and negative control, respectively. The activation was assessed according to formation of pronuclei, exocytosis of cortical granules and H1 kinase activity. The degree of DNA integrity in polocytes and M II oocytes was evaluated using a single cell gel electrophoresis (comet assay). Results: Continuous activation of oocytes with SNAP was associated with low developmental competence of parthenogenetic embryos. Repeated exposure to SNAP increased the activation efficiency. This corresponded with the results of comet assay. M II oocytes subjected to intermittent action of SNAP displayed a lower degree of DNA instability than those exposed to SNAP continuously. In comparison with M II oocytes, the polocytes exhibited a significantly higher level of DNA integrity. Nevertheless, the differences between the activation modes copied the trend observed in M II oocytes. Conclusions: (1) Intermittent exposure to SNAP provides a promissible approach to increase the efficiency of porcine oocytes activation in vitro. (2) Comet assay represents a suitable additional tool for evaluation of oocyte development quality. The project was supported by grant GACR 523/06/0295. doi:10.1016/j.reprotox.2008.05.035
doi:10.1016/j.reprotox.2008.05.034
RTX 6115 1–22
ETS Abstracts 2008 / Reproductive Toxicology xxx (2008) xxx–xxx
Apoptotic influence of cycloheximide an endocrine disruptor in lymphocytic organs during pregnancy
The effect of different activation modes on DNA integrity of porcine M II oocytes matured in vitro
E. Nikoloussis 1 , S. Massourides 2 , H. Frangou 2 , E.-N. EmmanouilNikoloussi 1
Bozena Novotna 1 , Marketa Sedmikova 2 , Jaroslav Kratochvilova 2 , Frantisek Jilek 2
1 Laboratory of Histology-Embryology and Anthropology, Faculty of Medicine, Aristotle University of Thessaloniki, Greece 2 Laboratory of General Biology, Faculty of Medicine, Aristotle University of Thessaloniki, Greece
1
Pregnancy and nutrition is a complex situation. Nutrients unfortunately come several times extracted from plants which have been recently vaporised with pesticides and fungicides. Cycloheximide [4-{(2R)-2-[(1S,3S,5S)-3,5-dimethyl-2oxocyclohexyl]-2-hydroxyethyl}piperidine-2,6-dione] and Molecular formula C15 H23 NO4 ; is a highly toxic agent and a protein synthesis inhibitor that acts specifically on the 60S subunit of eukaryotic ribosomes. It has been also been discussed as an endocrine disruptor as a common used fungicide. Although toxic, cycloheximide is a widely used fungicide, which must be very carefully applied due to diverse side effects possessing as a pesticide/infecticide drug for plants and fruits. The aim of this study was the Molecular and Transmission Electron Microscope analysis of lymphocytic organs of rats treated with cycloheximide during pregnancy. Cycloheximide (Sigma) was injected at 10 pregnant Wistar rats in dosage level of 3 mg/kg b.w. on 10th and 11th gestational days. Pregnancy on most of the rats resulted in abortions, absorptions and teratomas, while on two pregnant rats that were not sacrificed during pregnancy big breast tumors developed rapidly. Three months after the tumors were first observed, the animals were anesthetized the tumors were removed by a surgical operation and thereafter were sacrificed. Lymphocytic organs, i.e. spleen and lymph nodes were collected and proceeded for molecular and Transmission Electron Microscopical analysis to evaluate the role of the drug in apoptosis on spleen and lymph nodes. Animals’ spleen and lymph nodes as organs and tissues related to the tumors, were removed. For molecular evaluation of caspase-3 activity they were homogenized with a Polytron Kinematika homogenator in sucrose isotonic solution. Following subcellular fractionation according to the method of Nordlie and Lardy, the caspase-3 activity was determined in crude, nuclear, and cytosolic plus ribosomal fraction by a colorimetric assay (kit, Sigma), based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroaniline (pNA) moiety. Caspase-3 activity was calculated in mol pNA released per min per ml. Protein concentration was determined by the method of Bradford, modified by Bearden. Transmission Electron Microscopical analysis was performed under routine procedures for observations at Transmission Electron Microscope (TEM). Ultrastructural observations from spleen and lymph nodes cell parenchyma from pregnant rats showed severe pathological effects and increased apoptotic activity, accumulation of large secondary lysosomes and apoptotic bodies within cells.
Petr 3 , Jana
Institute of Experimental Medicine, CAS, v.v.i., Prague, Czech Republic University of Life Science, Prague 6, Czech Republic 3 Institute of Animal Production, v.v.i., Prague, Czech Republic 2
Introduction: Mammalian embryonic development requires an activating stimulus to overcome the block of oocytes at the stage of metaphase II. However, the physiological nature of this stimulus has not been fully clarified yet. This is one of the reasons why the procedures such as in vitro fertilization or cloning of mammals using the nuclear transfer are still characterized by a relatively low efficiency. Genomic integrity is also a prerequisite of successful development. We decided, therefore (1) to investigate the effect of different activation protocols on DNA of porcine oocytes matured in vitro using a nitric oxide donor (+)–S-nitroso-N-acetylpenicillamine (SNAP) as activating stimulus and (2) to relate the findings with the other parameters of developmental competence. Methods: The oocytes aspirated from ovaries of slaughtered gilts were cultured for 48 h in modified M 199 medium supplemented with gonadotropins. Then, the oocytes were devoid of cumulus cells and subjected to continuous (6 h) or repeated treatment with SNAP (10 min exposure to SNAP alternated with 10 min cultivation in free medium, the cycle was repeated 12 times and terminated by 2 h cultivation in free medium). Standard activation with calcium ions and culture without any treatment was used as positive and negative control, respectively. The activation was assessed according to formation of pronuclei, exocytosis of cortical granules and H1 kinase activity. The degree of DNA integrity in polocytes and M II oocytes was evaluated using a single cell gel electrophoresis (comet assay). Results: Continuous activation of oocytes with SNAP was associated with low developmental competence of parthenogenetic embryos. Repeated exposure to SNAP increased the activation efficiency. This corresponded with the results of comet assay. M II oocytes subjected to intermittent action of SNAP displayed a lower degree of DNA instability than those exposed to SNAP continuously. In comparison with M II oocytes, the polocytes exhibited a significantly higher level of DNA integrity. Nevertheless, the differences between the activation modes copied the trend observed in M II oocytes. Conclusions: (1) Intermittent exposure to SNAP provides a promissible approach to increase the efficiency of porcine oocytes activation in vitro. (2) Comet assay represents a suitable additional tool for evaluation of oocyte development quality. The project was supported by grant GACR 523/06/0295. doi:10.1016/j.reprotox.2008.05.035
doi:10.1016/j.reprotox.2008.05.034
RTX 6115 1–22