Figure 4.

Expression of MPF components and its activity is increased in the oocytes from aged females. (a) RT-PCR quantification of mRNA coding for CDK1 and B-type cyclins, as well as loading control Gapdh in the GV oocytes (0 h) from different age groups. For quantification of total RNA content in oocytes from YF and AF groups see Figure S3a. Values obtained for the YF group were set as 100%. Data was derived from at least four experiments of biologically different samples. Columns represent mean; error bars ± SD; ns non-significant; * p < 0.05, Student’s t-test. (b) Western blot analysis of CDK1, CDK1 (Thr161) and CCNB during oocyte maturation (0 h, 1 h and 6 h) in the both age groups. See Figure S3b,c for the assessment of global translation in oocytes from YF and AF groups. (c) Quantification of MPF components, CDK1, its phosphorylation (Thr161), CCNB and GAPDH as a loading control. Values obtained for the YF group were set as 100%. From at least three experiments of biologically different samples. Columns represent mean ± SD; * p < 0.05; ** p < 0.01; bars with ns are non-significant; Student’s t-test. (d) Representative image of analysis of CDK1 activity (H1) in the oocytes after isolation (0 h), NEBD (1 h) and at metaphase I (6 h). Kinase assay was done with oocytes of both female age groups. CDK1 activity was measured towards histone H1 as external substrate, marked by white rectangle. (e) Quantification of CDK1 (H1 substrate) activity during oocyte maturation from YF and AF groups. Measurements originated from four experiments of biologically different samples. Values obtained for the YF group were set as 100%. Columns represent mean; error bars ± SD; ns non-significant; * p < 0.05; Student’s t-test.