Notch signaling pathway promotes the development of ovine ovarian follicular granulosa cells.

ABSTRACT

The Notch signaling pathway regulates cell proliferation, differentiation and apoptosis involved in development of the organs and tissues such as nervous system, cartilage, lungs, kidneys and prostate as well as the ovarian follicles. This study aimed to investigate the mRNA expression and localization of NOTCH2, as the key factor in Notch signaling pathway. This was determined by PCR, real-time PCR and immunohistochemistry. Additionally, the effects of inhibiting Notch signaling pathway with different concentrations (5µM, 10µM and 20µM) of N-[N-(3, 5-Difuorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of Notch signaling pathway, on ovine granulosa cells was determined in vitro by detecting estradiol production using enzyme linked immunosorbent assay and expressions of the genes related to the cell cycle and apoptosis using real-time polymerase chain reaction (PCR). NOTCH2, the key member of Notch signaling pathway, was found in ovine follicles, and the expression of NOTCH2 mRNA was highest in the theca cells of the follicles in medium sizes (3-5mm in diameter) and granulosa cells of the follicles in large sizes (>5mm in diameter). Immunohistochemical results demonstrated that NOTCH2 protein was expressed in granulosa cells of preantral follicles, in both granulosa cells and theca cells of antral follicles. Compared with DAPT-treated groups, the control group had a higher number of granulosa cells (P<0.05) and a higher estradiol production (P<0.05). Compared with the control group, the mRNA abundances of HES1, MYC, BAX, BCL2 and CYP19A1 in DAPT-treated groups was lower (P<0.05), respectively; whereas, the expression of CCND2, CDKN1A and TP53 mRNA showed no remarkable difference compared with control group. Collectively, Notch signaling pathway could be involved in the ovine follicular development by regulating the growth and estradiol production of granulosa cells.