MicroRNAs transfected into granulosa cells may regulate oocyte meiotic competence during in vitro maturation of mouse follicles

Abstract

Do microRNAs (miRNAs) in granulosa cells (GCs) affect oocyte maturation during ovarian follicle development? Sophisticated regulation by miRNAs in ovarian GCs may improve oocyte maturation efficiency during ovarian follicle development. The meiotic competence of oocytes depends on the follicles potential to undergo appropriate maturation and is an important factor in infertility therapies such as IVF. The exact function of the GCs during follicular development remains unknown. After in vitro maturation (IVM) and ovulation induction of isolated ovarian pre-antral follicles from 12-day-old female C57BL6 mice (n 40), miRNA expression in the GCs was compared according to the maturity of the oocyte (metaphase I (MI) versus metaphase II (MII)). The miRNAs, which showed notable different expression, were modulated by transfection during IVM of follicles. miRNA expression and candidate target gene expression in GCs of isolated murine ovarian pre-antral follicles were evaluated by real-time PCR after IVM. miR mimics and -inhibitors for selected miRNAs were transfected into the in vitro-maturated follicles, and ovulation, oocyte maturation and fertilization rates were compared. Candidate target gene expressions in GC were evaluated by quantitative PCR and immunohistochemistry using confocal microscopy. The relative expression of mmu-let-7b (0.78 0.10, P 0.016), mmu-let-7c (0.78 0.12, P 0.029), mmu-miR-27a (0.57 0.18, P 0.016) and mmu-miR-322 (0.59 0.14, P 0.008) was significantly lower in the GCs of follicles containing MII oocytes compared with those of MI oocytes. Transfection with a mmu-miR-27a-mimic sequence decreased the oocyte maturation rate compared with that for the control (9.4 versus 18.9, P 0.042), and transfection with mmu-let-7c-, mmu-miR-27a- and mmu-miR-322-inhibitor sequences increased the oocyte maturation rate by 1.5- to 2.0-folds compared with that for the control (40.6, 31.6, and 30.5versus 18.9, P 0.001, P 0.013, P 0.021, respectively). The expression of IGFBP-2 was higher in GCs of MII than in the GCs of MI, and higher in miR-inhibitor transfection groups than in miR-mimic transfection groups and controls. An in vitro model was used in lieu of an in vivo model because of the ease of performing miRNA transfection in cell culture. However, studies have shown similarities and differences in in vivo versus in vitro cultured follicles. The findings of the present study need to be confirmed using in vivo maturation models and extended to evaluate developmental competence. Our findings suggest that sophisticated miRNA regulation in GCs may improve oocyte maturation efficiency during ovarian follicle development. This work was supported by a grant from the Korean Health Technology RD Project, Ministry of Health Welfare, Republic of Korea (A111539). None of the authors has any conflicts of interest to declare.