Chk1/2 inhibitor AZD7762 blocks the growth of preantral follicles by inducing apoptosis, suppressing proliferation, and interfering with the cell cycle in granulosa cells

Abstract

Background Checkpoint kinases 1/2 (Chk1/2) has an important role in somatic cells development and oocyte meiotic maturation. However, the role of Chk1/2 in folliculogenesis has not been fully elucidated. The aim of this study was to assess the effects of Chk1/2 inhibition on ovarian folliculogenesis and granulosa cells development in mice. Methods Preantral follicles (100 µm- 120 µm) and granulosa cells from pre-ovulatory follicles (pre-GCs) of mice were isolated and cultured with or without Chk1/2 inhibitor AZD7762. Preantral follicles were cultured for 96 h. Then, follicle morphology and follicular growth were assessed every 48 h. Granulosa cells were cultured for 48 h with or without AZD7762, after which cell apoptosis, cell proliferation, and cell cycle analysis were assessed; meanwhile, the mRNA expression of PCNA and Bax were measured by real-time RT-PCR, and PCNA and Bax protein were measured by Western blot. Results Compared with control follicles, AZD7762 inhibited growth of preantral follicles. Furthermore, inhibition of Chk1/2 significantly induced apoptosis and inhibited the proliferation of granulosa cells, arrested cell cycle at S and G2/M phases, and decreased G1 phase fraction. Also, the expression of PCNA mRNA and protein were significantly reduced, while Bax mRNA and protein were significantly increased post AZD7762 treatment in granulosa cells. Conclusions This study revealed that Chk1 and Chk2 have a crucial role during preantral follicular development by regulating the proliferation and apoptosis of granulosa cells.