Fas ligand expression and mediated activation of an apoptosis program in bovine follicular granulosa cells

摘要

Fas ligand (FasL) is a cytokine that may be expressed as a transmembrane ligand at the cell surface, and induces apoptosis by binding to the Fas. Ovarian follicular atresia and luteolysis are thought to occur by apoptosis. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, FasL gene without the stop codon was amplified and directly cloned into pAcGFP-N1. The resultant recombinant plasmid pAcGFP-bFasL was then transfected into bovine follicular granulosa cells. The transcription and translation of FasL were detected by RT-PCR and Western blot analysis. The methyl-tetrazolium (MTT) assay, Hoechst33342 staining, and DNA Ladder method were performed to determine the growth inhibition and apoptosis of the cells. The real-time quantitative PCR assay was performed to measure the expression of FasL in vivo in granulosa cells collected from diverse stage of dominant and atretic follicles. The results showed that the FasL fusion gene was successfully expressed in granulosa cells as evidenced by the detection of a 847. bp fragment corresponding to the FasL mRNA by RT-PCR and a 59. kDa band corresponding to the FasL fusion protein by Western blot. Granulosa cell viability decreased significantly at 72. h after transfection, and the apoptosis rate of the cells transfected with pAcGFP-FasL was significantly higher than that of the control group. Cells in the FasL transfection group showed ladder patterns characteristic of apoptosis, and the nuclei were shrunken and densely hyperchromatic or fragmented. In addition, FasL was highly expressed in granulosa cells of atretic follicle than dominant follicle in vivo. We found that FasL is capable of inhibiting the proliferation of bovine follicular granulosa cells and inducing cell apoptosis in vitro and in vivo when over-expressed. This study will aid in further understanding the mechanism of regulation of FasL on bovine oocyte formation and development.