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Fig. 4. Degradation of NO145 protein during oocyte maturation by the proteasome system. (A) Gradual disappearance of endogenous NO145 protein during oocyte maturation. Defolliculated stage VI oocytes were treated with progesterone (P; after 2 hours). Mature oocytes were collected at the indicated times (M_0-120; in minutes). Lanes VI and E indicate immature stage VI oocytes and eggs, respectively. An amount equivalent to one oocyte per lane was loaded and immunoblotted with antibodies against NO145 and NO38. Analogous oocyte stages were analyzed by the RACE-PAT method. (B) Phosphorylation of NO145 in maturing oocytes. Lysates from stage VI oocytes, matured oocytes (M₀) and eggs (E), were treated with calf intestine alkaline phosphatase (CIP) as indicated and analyzed by immunoblotting with antibodies against NO145 and NO38. The positions of fast- and slow-migrating protein variants are indicated by close and open arrowheads, respectively. (C) Schematic representation of the experimental protocol to analyze the involvement of the proteasome pathway in the degradation of protein NO145. (D) Methylated ubiquitin affects the degradation process of NO145 during maturation. Methylated ubiquitin or injection buffer were injected into oocytes at GVBD₅₀. Samples were taken 0, 30, 60, 120 and 180 minutes after appearance of the white spot (M₀-M₁₈₀) and protein extracts were analyzed by immunoblotting using NO145 antibodies. (E) Degradation of the endogenous NO145 protein is blocked by proteasome inhibitors. Immature stage VI oocytes (VI) were induced to mature by addition of progesterone. At GVBD₅₀ oocytes with (+) or without (–) a white spot were taken as controls. At this time, non-white spot oocytes were injected with either MG132, lactacystin or injection buffer alone (lanes indicated by +) or left uninjected (lanes indicated by –). All oocytes were incubated until GVBD₁₀₀ or overnight (E) and crude extracts from five oocytes each were prepared, and the equivalent of one oocyte or egg per lane was separated by SDS-PAGE. The resulting immunoblot was probed with antibodies against NO145 and finally re-probed with Xp54 antibodies as loading control.

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