CHARACTERIZATION OF THE DE-NOVO PROTEIN-S YNTHESIS IN THE EARLY STAGES OF DEVELOPMENT OF PORCINE EMBRYOS COLLECTED IN-VIVO AND CULTURED IN NCSU-23 MEDIUM

Embryo development following supplementation of media with taurine and hypotaurine, and the influence of additional BSA-application, was inv estigated by assessing embryonic protein synthesis. In vivo developed and in vitro cultured zygotes (Z), 2-, 4-cell stage embryos, morulae ( Mo) and blastocysts (Bl) were used. Five embryos per group were labell ed (4 h) in 50 mu l drops of NCSU-23 or modified NCSU-23 containing 1 mCi/ml of [S-35]-methionine (> 1000 Ci/mM, Amersham Corp., Braunschwei g, Germany), successively rinsed with non-radioactive medium and place d in 60 mu l SDS-lysis buffer. The same counts per minutes (cpm) of ea ch embryonic stage were used for 1-D electrophoresis (Tris/Tricine buf fer system). Gels were pretreated with fluorite (Amplify, Amersham Cor p.), dried, and exposed to Hyperfilm MP (Amersham Corp.) at -70 degree s C. The profiles for incorporation of radiolabelled methionine into p rotein were different according to developmental stages, to in vivo de veloped and in vitro cultured embryos, as well as to cultivated embryo s with and without BSA. The incorporation of [S-35]-methionine into pr otein decreased from the time of fertilization to the 4-cell stage and increased at the blastocyst stage. In vivo developed blastocysts inco rporated more methionine than in vitro cultured blastocysts. Incorpora tion into in vitro blastocysts, which were cultivated without BSA, was decreased in comparison to in vivo and in vitro stages cultivated wit h BSA. Qualitative changes in protein profiles depended on the develop mental stage of the embryos and were similar to in vivo and in vitro s tages. The changes in protein profiles were observed between Z and 2-c ell embryos (dominant bands at 97, 67, 45, 34, 20, 17, 6 kd) compared to 4-cell embryos (bands at 30, 20 kd were absent, new band at 47 kd) and blastocysts (different to other cell stages: four bands at 45-60 k d). Only small differences in protein pattern were found between embry os incubated with taurine and hypotaurine and in vivo cell stages, res pectively. The lack of BSA had no influence on the de novo synthesized proteins. The results indicate that supplementation of culture media with taurine and hypotaurine in combination with BSA beneficially infl uenced embryonic protein metabolism and development. However, the decr eased incorporation of methionine into in vitro blastocysts demonstrat ed that cultivation of embryos over a longer period has yet to be subs tantially improved.