Infertility of the GDF9-Ccnb1^−/− female mice. (A) Construction of Ccnb1^Flox/Flox and generation of Ccnb1^Flox/Flox;GDF9-Cre mice. Exons 5–9 of Ccnb1 were deleted by GDF-9-Cre–mediated recombination. (B) Genotyping PCR for the identification of the GDF9-Ccnb1^−/− mice. The Flox band was 673 bp, and the WT band was 475 bp. (C) Morphology of the GDF9-Ccnb1^−/− female mouse. The size of the GDF9-Ccnb1^−/− female was comparable with the control female. (D) Real-time PCR showing the loss of Ccnb1 mRNA in the GDF9-Ccnb1^−/− mouse oocytes. 7-wk-old control and GDF9-Ccnb1^−/− mice were used. Gapdh was used as the internal reference, and the mRNA expression of the control group was normalized to 1. ***, P < 0.0001. (E) Western blot demonstrating the absence of Cyclin B1 protein in the GDF9-Ccnb1^−/− mouse oocytes. A total of 150 oocytes that underwent GVBD (after 2 h incubation in M2 medium at 37°C) were used in each lane, and the level of GAPDH was used as an internal control. The experiment was repeated three times. (F) Fertility curves of the GDF9-Ccnb1^−/− female mice (n = 7; magenta line) and the control females (n = 7; green line).