Regulative Relationship Between CDK2AP1and Genes Related To Chicken Embryonic And Gonadal Development

Gonadal differentiation and development of the animal has been a hotspot for researchers, and have important significance for the biological basis studies and treatment of reproductive diseases. SRY, the mammalian sex switch gene has been found, but poultry sex determination and gonadal development regulation mechanism is unclear. So it is necessary to clarify the candidate genes expression characteristics of poultry gonadal development and related functions. During early development of chicken embryo gonadal, our laboratory has screened a differential expression gene, CDK2AP1, between female and male individuals. Studies in mouse have found that it plays an important role in gonadal development. Therefore, this experiment will take advantage of over-expression and RNA interference technology, analyze the regulatory relations between chicken sex determination and differentiation related genes and CDK2AP1. We have initially identified its function during the chick embryo gonadal developing process. The main studies are as follows:1. The expression of CDK2AP1was detected in the3.5d (St22) and4.5d (St25) chick embryo, and5.5d (St28) and6.5d (St30) genitourinary system by whole-mount in situ hybridization. The results showed that it expressed in a variety of embryo tissues at3.5d (St22) and4.5d(St25), particularly higher in the head and limbs. CDK2AP1expression level in female gonads is significant higher than in the male and at6.5d (St30) but not significant at5.5d (St28). The scope and intensity of expression in male and female gonads at5.5d (St28) are higher than that at6.5d (St30)2. The over-expression retrovirus vectors of CDK2AP1, RCASBP.B-CDK2AP1-GFP (CGR) and three RNA interference retrovirus vectors of CDK2AP1, RCASBP.B-U6-shRNA-GFP (UCR1, UCR2and UCR3) were constructed using gene recombination technique. The result of qPCR and WB show that CDK2AP1expression levels increased significantly in the over-expression group and decreased significantly in UCR1and UCR3RNA interference group (P<0.01), no difference in UCR2.3.Analyzing result of gene regulatory relations between CDK2AP1and gonadal development and differentiation associated genes, it showed that CDK2AP1could significantly up-regulate AR, ERa and FOXO3a expression levels (P<0.01), significantly down-regulate CDK2(P<0.01) and TWIST2expression levels was no difference.We can initially identified CDK2AP1plays an important role in sexually differentiation and gonadal development through regulating related gene AR, ERa and FOXO3a expression levels.4.Test results of flow cytometer displayed that the over-expression CDK2AP1can significantly increase the proportion of cells in G1phase (P<0.01), significantly reduce the proportion of cells in S and G2/M phase (P<0.01); during RNA interference group, the proportion of cells in G1phase decreased significantly (P<0.01), no difference that in S-phase (P>0.05), G2/M of cell ratio increased significantly (P<0.01).