Authors : Run-Jun Yang, Jun-Ya Li, Xue Gao, Zhi-Hui Zhao, Lu-Pei Zhang, Hui-Jiang Gao and Shang-Zhong Xu
Abstract: Fas-Associated Death Domain (FADD) is a signal connection protein in the Fas/FasL system which may play a key role in apoptosis. Follicle 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. This was accomplished by cloning the FADD gene using RT-PCR, deleting the termination codon in its cDNA and directionally cloning the amplified FADD gene into the eukaryotic expression vector pAcGFP-N1. The pAcGFP-bFADD recombinant plasmid was then transfected into bovine follicular granulosa cells by using lipofectamine 2000. Expression of AcGFP was observed under fluorescent microscopy and the transcription and expression of FADD was detected by RT-PCR and Western-blot. Then the Methyl-Tetrazolium (MTT) assay was performed to determine the growth inhibition of cells. Cell apoptosis was observed by using Hoechst33342 staining and DNA-Ladder Method. The results showed that the pAcGFP-bFADD recombinant plasmid was successfully constructed. AcGFP expression was detected as early as 24 h after transfection. A 654 bp fragment was amplified by RT-PCR and a 51.4 kD target protein was detected by Western-blot. Cells viability decreased significantly at 72 h after transfection and the apoptosis rate of the cells transfected with pAcGFP-FADD was significantly higher than the control group. Cells in the FADD transfection group showed ladder patterns characteristic of apoptosis and the nuclei were shrunken and densely hyperchromatic or fragmented suggesting that FADD is capable of inhibiting the proliferation of bovine follicular granulosa cells and inducing cell apoptosis when over-expressed.
Run-Jun Yang, Jun-Ya Li, Xue Gao, Zhi-Hui Zhao, Lu-Pei Zhang, Hui-Jiang Gao and Shang-Zhong Xu, 2011. Fas-Associated Death Domain (FADD) Mediated Activation of a Apoptosis Program in Bovine Follicular Granulosa Cells. Journal of Animal and Veterinary Advances, 10: 1994-2003.