Fig 2: Expression of CSN3 and CSN5 mRNA in oocytes and follicular cells.(A) Expression of CSN3 and CSN5 mRNA in oocytes during in vitro maturation. For semi-quantitative PCR, single oocyte-equivalent cDNA was used as a template for amplification. GV, GVBD, MI, and MII oocytes were harvested at 0, 2, 8, and 16 h, respectively. Constitutively expressed oocyte-specific H1foo was used as an internal control and synthetic GFP mRNA was added to the lysates before mRNA isolation to compare the efficiency of mRNA recovery. (B) Expression of CSN3 and CSN5 mRNA in follicular components. Oo, oocytes at GV stage; CC, cumulus cells; GC, granulosa cells. Gdf9 and Fshr were used as markers for oocytes and granulosa cells, respectively.

Fig 3: The level of APC/C substrates changed following CSN3 or CSN5 knockdown.(A) Western blot analysis of Cdc2, Cyclin B1, and Securin by using 100 oocytes. Numbers on the left side of the band indicate the sizes (kDa) of the protein markers, while arrows indicate the specific proteins. (B) Western blot analysis of Cdc2, Cyclin B1, and Securin by using 100 oocytes. (C) Relative protein level of each band was calculated and presented as the mean ± SEM by measuring the area and data are presented in comparison to appropriate control bands for each protein. Asterisks indicate statistically significant differences compared to control group (p<0.05).

Fig 4: A schematic diagram depicting the proposed role of CSN in the control of meiotic chromosome segregation in oocytes based on loss-of-function of CSN3 or CSN5.(A) CSN regulates APC/C activation, followed by degradation of Cyclin B1 and Securin. Degradation of Cyclin B1 induces MPF inactivation, while degradation of Securin induces activation of Separase that brings Cohesin cleavage and chromosome separation. Following these sequences, meiosis I is completed and first polar body is extruded. (B) When normal CSN is absent, APC/C is not activated, and degradation of Cyclin B1 and Securin are also failed. Maintained MPF activity and intact Securin resulted is maintained high, cohesions are intact, so chromosomes cannot be separated. And then meiosis I is failed; the oocytes are arrested at MI stage.

Fig 5: Dual kinase activity assay after CSN3 or CSN5 RNAi.MPF and MAPK activities were assessed by measuring the level of phosphorylation of Histone H1, a substrate of MPF, and myelin basic protein, a substrate of MAPK. Oocytes were collected every 2 h during in vitro maturation. Lower panel, relative MPF activity was determined by quantifying phosphorylation of substrates and was expressed relative to that of control oocyte at 2 h.