Effects of different kinases and phosphatases on nuclear and cytoplasmic maturation of bovine oocytes

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Abstract
Phosphorylation is considered as a common post‐translational modification implicated in the control of various key enzymes. In somatic and germinal cells, important regulators of the cell cycle are controlled by their phosphorylation status, and some act as kinases or phosphatases themselves. Bovine oocytes are blocked in the germinal vesicle (GV) stage until either an LH surge occurs or until oocytes are released from the inhibitory influence of the follicle. Meiotic resumption in vitro is therefore an excellent model for the study of phosphorylation events that occur in the G2/M transition, a control point of the cellular cycle. To better understand this transition, we have modulated, either directly or indirectly, kinases using known effectors (epidermal growth factor, EGF; isobutyl‐methylxanthine‐forskolin, Bx‐Fk; 6‐dimethylaminopurine, 6‐DMAP) or phosphatases (okadaic acid, OA) or cycloheximide, which is known to inhibit maturation through protein synthesis suppression. With this procedure, influence on meiotic resumption and phosphoprotein patterns was verified. Both EGF and OA accelerated nuclear maturation after 9 hr of culture. Only 23% (n = 140) and 9% (n = 111) of oocytes were still at GV stage with EGF and OA, respectively, compared to 41% (n = 105) of control oocytes. The different treatments changed the protein patterns in oocytes. In cumulus cells, the patterns were especially modified by the OA treatment. Characteristic changes that occur in germ cells were also identified. Nuclear maturation was inhibited by modulators of kinase (6‐DMAP, GV = 74%, n = 126; cAMP dependent protein kinase (PKA) stimulators, Bx‐Fk, GV = 71%, n = 129) likewise, phosphoprotein patterns were affected, especially in oocytes. The cycloheximide treatment was able to maintain nearly all oocytes in GV after 9 hr of culture (GV = 92%, n = 131). This analysis allowed the identification of substrates for the different effectors used in this study and also helped in determining the levels of phosphorylation required for nuclear maturation.