Fertilization and Early Embryogenesis
(W339) ACTIVATION OF PROTEIN KINASE C AND SUBSEQUENT INHIBITION OF PROTEIN PHOSPHORYLATION IMPROVE EMBRYONIC DEVELOPMENT OF BOVINE OOCYTES AFTER ICSI.
Suh, Tae Kwang1, Seidel, George1, 1 Colorado State University, Fort Collins, CO
ABSTRACT- Activation of protein kinase C (PKC) and dephosphorylation/inactivation of kinases are involved in the transition of metaphase to interphase after fertilization. In this study, we studied the effect of the PKC activator, phorbol 12-myristate 13-acetate (PMA), and subsequent treatment with the protein serine/threonine kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on embryonic development of bovine oocytes in vitro after intracytoplasmic sperm injection (ICSI). A 3x2 factorial experiment consisting of 3 PMA (0, 0.01, 0.02 M) and 2 6-DMAP (0, 2mM) concentrations was conducted. Semen from 3 bulls were used twice to produce embryos in 6 replicates. Chemically defined media (CDM) were used throughout. Oocytes obtained from slaughterhouse ovaries were matured in vitro in CDM-M for 22-22.5h under 5% CO2 in air at 38.5oC, and oocytes with a 1st polar body were used for ICSI. Motile sperm from frozen-thawed semen were used for sperm injection with a Piezo driven pipet. After ICSI, presumptive zygotes were cultured in CDM-1 containing PMA for 2h, and then 6-DMAP for 3h according to the design. Zygotes were cultured in CDM-1 for 2.5 days, and then in CDM-2 for 5 days at 39oC in 5% CO2/ 5% O2/ 90% N2. Data were subjected to ANOVA; the arc sin transformation was used for percentage data for each replicate. Cleavage in 0, 0.01, and 0.02 M PMA groups, in the absence of 6-DMAP, was 6.7, 8.3, 14.2%, respectively, and in its presence, was 26.5, 36.2, and 49.4%, respectively (P<0.05). Respective blastocyst rates per oocyte were 0, 0, 5.8, 5.7, 10.7, 11.7% (P<0.05). This study showed that activation of protein kinase C plus subsequent inactivation of protein phosphorylation improves embryonic development of bovine oocytes after ICSI.
KEY WORDS: PKC, phosphorylation, bovine, ICSI