The Mining And Functional Study Of The Candidate Genes For Asthenospermia Of The Rooster

In poultry industry, around 5 to 12% breeder roosters were eliminated because of dyszoospermia. The reproductive efficiency of indigenous breed was also significantly lower than the standard commercial breeds, which impeded the reproduction and breeding system and industrialization development.To explore the pathogenesis of the rooster asthenospermia, the expression profile of six candidate genes (COX7B, PTGDS, hPGDS, SPAG6, WNT2, and CCNF), these genes were selected from the previous digital gene expression profiling (DGE) and bioinformation analysis. A total of 300 Beijing-You roosters of 42 weeks were observed for 4 weeks for semen quality including semen volume, sperm mobility, etc. Fifteen asthenospermia and 15 normal birds were selected accordingly. Real-time quantitative PCR were performed to determine the expression of the six genes in the testicular tissue of those birds.The gene CCNF was selected for further functional study. The mRNA expression of CCNF was firstly determined in SSCs, sertoli cell and DF1 cell line. Four lentiviral vector harboring RNAi sequence targeting CCNF was constructed. The RNAi sequence of highest efficiency was selected for interfering the CCNF expression in sertoli cell and DF1 cell line. At 24,48,72, and 96 h after transfection, the expression of CCNF were determined using RT-qPCR, the cell proliferation was measured using CCK8. The results indicated that:1. COX7B and PTGDS were significantly high-expressed in the asthenospermia group (P<0.01), while the WNT2, hPGDS, and CCNF were significantly low-expressed in the asthenospermia group (P<0.05). However, the expression of SPAG6 gene was not significantly different in the two groups. These results were in accordance with the previous DGE analysis. The expression patterns of the six genes identified from DGE for asthenospermia in chickens were validated in the present study. They can be important candidate genes for further functional study and revealing the mechanism of chicken asthenospermia.2. The expression of CCNF can be detected in SSCs, sertoli cell and DF1 cell line. There was no significant difference in expression of CCNF in three cells.3. Optimum condition that was the best for the transfection of RNAi sequence into DF1 and sertoli cell was:virus titer 5×106 TU/mL and MOI=5. Transfection efficiency was 60%, where only 20% in SSCs. At 72 and 96 h after transfection of siR-1, the expression of CCNF siR-1 was significantly decreased (P<0.01), indicating that the lentiviral vector harboring RNAi sequence targeting CCNF was efficient.4. At 72 and 96 h after siR-1 transfection, the cell proliferation of DF1 and sertoli cell was significantly decreased than the control group (P=0.05 and 0.01, respectively). This indicated that the CCNF may be involved in the regulation of cell proliferation...