The Preliminary Study Of AQP9 Gene Expression And Its Relationship With Apoptosis In Buffalo Granulosa Cells

Aquaporins (AQPs) are kinds of specific water-selective membrane channel proteins, which widely exist in prokaryotic and eukaryotic cell membrane. Based on studies of mice and human, it was found that, besides the function of transporting water, some of AQPs involved in the formation and development of follicles, granulosa cell apoptosis, follicular atresia and the fertility of female animal, but the mechanism was still unclear. In present study, we first cloned the buffalo AQP9 gene, then analyzed its expression pattern in buffalo follicules and during early embryonic development process, in addition the expression of AQP9 and apoptosis-related genes in granulosa cells from buffalo follicles with different atretic status and oocyte cumulus cells matured in vitro. Then, we explored the relationship between the expression of AQP9 gene and the apoptosis of buffalo granulosa cells, and the role of AQP9 in granulosa cell apoptosis during PKC signaling pathway by over-expressed AQP9 and adding PKC inhibitor staurosporine in buffalo granulosa cells. The research can lay the foundation for understanding follicular atresia mechanism and improving buffalo reproductive performance. The main results were as following:Firstly,888 bp buffalo AQP9 gene coding sequence was got by RT-PCR method, it predicted encoding 295 amino acids. Results of multiple sequence comparison showed that the nucleotide sequence of buffalo AQP9 shared 99%? 90%?97% and 88% homologeous compared with that of Bos Taurus, Sus scrofa, Ovis ariessis and Homo sapiens, respectively; while shared 99%?86%?97%? 83% homologeous for amino acids, respectively. Results of phylogenetic tree analysis indicated that AQP9 gene was highly conservative in the evolutionary process. Protein structure analysis showed that buffalo AQP9 protein had 17 threonine (Thr), did not contain the signal peptide, it had potential phosphorylation sites and a specific Pfam MIP domain.Secondly, research on the expression of AQP9 in buffalo female reproductive cells found that AQP9 protein was expressed in buffalo granulosa cells from primordial follicles, primary follicles, secondary follicles to antral follicles. qRT-PCR results showed that, compared with GV oocytes, the expression of AQP9 gene in M? oocytes matured in vitro was significantly higher (p<0.05). Its expression was not detected at 2-cell,4-cell and the morula stages, but appeared in blastocysts. In granulosa cells from buffalo follicles with different atretic status, the expression of AQP9 and apoptosis-related genes Bax, caspase3 and Fas had a increasing trend with the develepment of follicular atresia. The expression of AQP9 gene increased significantly in early stage atretic follicles (p<0.05). In cumulus cells with different maturation degrees in vitro, the expression of AQP9 in good matured granulosa cells increased significantly (p<0.05) compared with that of bad matured; meanwhile, the expression of apoptosis-related genes Bax, caspase3, Fas increased significantly (p<0.05).Lastly, the study on buffalo granulosa cell apoptosis and AQP9 expression found that, when serum-free cultured for 36 h after transfected buffalo granulosa cells with AQP9 over-expression vector, apoptosis-related genes Bax, caspase3 and Fas increased significantly (p<0.05), Bcl-2 downregulated significantly (p<0.05). In the study of the relationship between the buffalo granulosa cell apoptosis and AQP9 gene expression mediated by PKC inhibitor staurosporine found that after treated with different concentration staurosporine, the morphology of buffalo granulosa cells became collapse, cell membrane appeared drawing formed dispersing to surrounding, cytoplasmic vacuoles increased, the suspended death cells increased. Flow analysis results showed staurosporine can promote granulosa cell apoptosis with a concentration dependent manner., The expression of AQP9 and caspase3, Bax, p53 genes upregulated significantly with the deepening of apoptosis degree (p<0.05). The expression of AQP9 and Bax protein also increased with the increasing of apoptosis.The above results demonstrated that AQP9 gene was highly conservative among different species. It involved in buffalo follicle development, oocytes maturation, early embryo development and follicular atresai. Its expression was closely related to buffalo granulosa cell apoptosis and maybe had effect on oocytes maturation by influencing apoptosis of cumulus cells. AQP9 may play an important role in the apoptosis of buffalo granulosa cells mediated by PKC signaling pathway.