The amount of cAMP in porcine oocytes cultured with olomoucine

Using the enzyme-linked immunosorbent assay and the method of reverse transcription-polymerase chain reaction, cAMP and adenylate cyclase mRNA were demonstrated in porcine oocytes with cumulus cells (COCs) and denuded oocytes (DOs) treated with olomoucine. an inhibitor of cyclin dependent kinase of maturation promoting factor, and the cAMP content and the ability to synthesize cAMP in the treated oocytes and cumulus cells were examined. The fluctuation of cAMP content and the ability to synthesize cAMP in porcine oocytes and cumulus cells cultured for various periods were also examined. The concentration of cAMP in olomoucine-treated COCs was 173 fM, which was significantly lower than 3.067 fM in non-treated COCs. whereas the concentration of cAMP in DOs treated with olomoucine (39 fM) was similar to that in non-treated DOs (24 fM). The concentration of cAMP in cultured DOs was always low and did not significantly change during maturation culture. The concentration of cAMP in cultured COCs was low until 8 hrs of culture, whereas that increased rapidly after 8 hrs of culture and reached the maximum at 12 hrs of culture. The concentration of cAMP in COCs decreased after 22 hrs of culture. The expression of adenylate cyclase mRNA was weaker in COCs and DOs treated with olomoucine than in control COCs and DOs. In cultured COCs and DOs, the expression of such the mRNA was always demonstrated and stronger in those cultured for 8 hrs than in those cultured for 0, 22, 32 and 44 hrs. From these results, it was suggested that cAMP contained in COCs is mostly derived from cumulus cells, although cAMP is synthesized in oocytes. The present findings also suggested that the olomoucine treatment inhibits cAMP synthesis in cumulus cells, which in turn results in the low cAMP level in the cytoplasm of oocytes.