Effects of Notch2 on proliferation, apoptosis and steroidogenesis in bovine luteinized granulosa cells

Abstract

Notch signaling pathway plays an important regulatory role in the development of mammalian follicles. This study aimed to explore the effect of Notch2 on the function of bovine follicles luteinized granulosa cells (LGCs). We detected that the coding sequence (CDS) of bovine Notch2 gene is 7416 bp, encoding 2471 amino acids (AA). The homology of Notch2 AA sequence between bovine and other species is 86.04%–98.75%, indicating high conservatism. Immunohistochemistry found that Notch2 receptor and its ligand Jagged2 localize in granulosa cells (GCs) and theca cells in bovine antral follicles. And immunofluorescence found that positive signals of Notch2 and Jagged2 overlap in bovine LGCs, speculating that Notch2 receptor may react with Jagged2 ligand to activate Notch signaling pathway and play an important role in bovine LGCs. To further investigate the function of Notch2, Notch2 gene was silenced by short hairpin RNA (shRNA) and CCK-8 analysis showed that the proliferation rate of LGCs was downregulated significantly (P < 0.01). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that the mRNA expression of apoptosis related gene Bcl-2/Bax decreased (P < 0.01) and Caspase3 increased (P < 0.05), cell cycle related gene CyclinD2/CDK4 complex decreased (P < 0.01) and P21 increased (P < 0.05), steroidogenesis gene STAR and 3β-HSD decreased (P < 0.01) while CYP19A1 and CYP11A1 had no significant difference (P > 0.05). In addition, Enzyme-linked immunosorbent assay (ELISA) showed that there was no difference in estradiol (E2) secretion (P > 0.05) while the progesterone (P4) secretion decreased (P < 0.01). In conclusion, Notch2 plays an important role in regulating bovine LGCs development.