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) have an important role in somatic cell 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 cell development in mice. Methods. Preantral follicles (100-120 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 96h. Then, follicle morphology and follicular growth were assessed every 48h. Granulosa cells were cultured for 48h 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 (P<0.05). Furthermore, inhibition of Chk1/2 significantly induced apoptosis (P<0.05) and inhibited the proliferation of granulosa cells (P<0.01), arrested cell cycle at S and G2/M phases, and decreased G1 phase fraction (P<0.001). Also, the expression of PCNA mRNA and protein were reduced (P<0.01), while Bax mRNA and protein were increased (P<0.05) 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.