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Effect of co-culture cells on the uptake of cysttne into bovine embryos
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Theriogenology
EFFECT OF CO-CULTURE CELLS ON THE UPTAKE OF CYSTINE JNTO BOVINE EMBRYOS M.Takahashil,T. Nagai2, N. Okamura3 and A. Okanol IDepartment of Animal Reproduction, National Institute of Animal Industry, Tsukuba 305 Japan ; 2Livestock Division, Tohoku National Agricultural Experiment Station, Morioka Japan ; 3Division of Biochemistry, Tsukuba University School of Medicine, Ibaraki 305 Japan. We have reported that intracellular glutathione is related to the development of bovine IVM-IVF embryos. Although glutathione synthesis is highly influenced by extra cysteine(Cys), Cys is easily oxidized to cystine(CyssCy). Some kinds of cells such as lymphocytes can not utilize CyssCy. However, when they are co-cultured with feeder cells, they can proliferate by utilizing Cys reduced from CyssCy by feeder cells. We investigated the effect of co-culture cells on the uptake of CyssCy into bovine embryos. Bovine 6-8 cell embryos obtained by JVM-IVF oocytes were cultured for 0, 1,2, 3 and 4 h in Cys, CyssCy -free TCM-199 supplemented with r4C-CyssCy with or without cumulus-granulosa cells, previously collected and cultured at a concentration of 5x105 cells/dish for 2 or 3 d. Embryos were washed 6 times in Ca2+ and Mg2+ free Phosphate buffered saline used for the PBS supplemented with 2 mg/ml PVP. embryo washing was collected and used for background counting. After embryos were lysed, incorporated radioactivity was measured by using scintillation counter. Incorporated radioactivity into co-culture cells was also treated as described above. Culture medium was collected and analyzed by using HPLC system with radioisotope detector. incorporation of *4C-CyssCy into embryos cultured for 1,2,3 and 4 h without co-culture cells was 5.921.7, 11.724.1, 8.822.4 and 12.023.9 cpm/embryo, respectively. In contrast, incorporated radioactivity into embryos cultured with coculture cells was 7.6kO.5, 14.3~3.1, 22.4k4.7 and 36.6=5.5 cpm/embryo, respectively. Incorporated radioactivity of co-culture cells increased after a lapse of culture (26435717 to 7502k1206 cpm/5xl@ cells). Subsequently t4C-CyssCy in the medium with co-culture cells decreased however, it was detected in other components. These results indicate that 1)bovine 6 to 8-cell embryos can hardly utilize cystine without coculture cells, 2) cystine in the medium is changed to other components through coculture cells and secreted to culture medium, and 3) 6 to 8-cell embryos can utilize the components derived from cystine .
Theriogenology
EFFECT OF CO-CULTURE CELLS ON THE UPTAKE OF CYSTINE JNTO BOVINE EMBRYOS M.Takahashil,T. Nagai2, N. Okamura3 and A. Okanol IDepartment of Animal Reproduction, National Institute of Animal Industry, Tsukuba 305 Japan ; 2Livestock Division, Tohoku National Agricultural Experiment Station, Morioka Japan ; 3Division of Biochemistry, Tsukuba University School of Medicine, Ibaraki 305 Japan. We have reported that intracellular glutathione is related to the development of bovine IVM-IVF embryos. Although glutathione synthesis is highly influenced by extra cysteine(Cys), Cys is easily oxidized to cystine(CyssCy). Some kinds of cells such as lymphocytes can not utilize CyssCy. However, when they are co-cultured with feeder cells, they can proliferate by utilizing Cys reduced from CyssCy by feeder cells. We investigated the effect of co-culture cells on the uptake of CyssCy into bovine embryos. Bovine 6-8 cell embryos obtained by JVM-IVF oocytes were cultured for 0, 1,2, 3 and 4 h in Cys, CyssCy -free TCM-199 supplemented with r4C-CyssCy with or without cumulus-granulosa cells, previously collected and cultured at a concentration of 5x105 cells/dish for 2 or 3 d. Embryos were washed 6 times in Ca2+ and Mg2+ free Phosphate buffered saline used for the PBS supplemented with 2 mg/ml PVP. embryo washing was collected and used for background counting. After embryos were lysed, incorporated radioactivity was measured by using scintillation counter. Incorporated radioactivity into co-culture cells was also treated as described above. Culture medium was collected and analyzed by using HPLC system with radioisotope detector. incorporation of *4C-CyssCy into embryos cultured for 1,2,3 and 4 h without co-culture cells was 5.921.7, 11.724.1, 8.822.4 and 12.023.9 cpm/embryo, respectively. In contrast, incorporated radioactivity into embryos cultured with coculture cells was 7.6kO.5, 14.3~3.1, 22.4k4.7 and 36.6=5.5 cpm/embryo, respectively. Incorporated radioactivity of co-culture cells increased after a lapse of culture (26435717 to 7502k1206 cpm/5xl@ cells). Subsequently t4C-CyssCy in the medium with co-culture cells decreased however, it was detected in other components. These results indicate that 1)bovine 6 to 8-cell embryos can hardly utilize cystine without coculture cells, 2) cystine in the medium is changed to other components through coculture cells and secreted to culture medium, and 3) 6 to 8-cell embryos can utilize the components derived from cystine .