Study The Regulatory Function Of MiR-2954 In The Gonadal Sex Differentiation Of Chicken Embryos By Using Vivo-morpholinos And Sex Reversal Model

The sex determination and gonadal sex differentiation of poultry are regulated by a variety of factors.During this process,the regulatory function of some genes,such as testicular determinants DMRT1 and ovarian development-related gene FOXL2,have been well studied.The specific expression of miRNA in some tissues is helpful to study its function on the development of embryonic organs.At present,it has been found that multiple miRNAs exhibit sex-biased expression in avian gonads and have the potential function on regulating the gonadal differentiation.Our research group previously has screened a sex-biased expressed miRNA,miR-2954,which has higher expression in chicken embryonic gonads and other tissues.On the other hand,miR-2954 regulates the expression of male sex-related genes DMRT1 and SOX9 through target genes YY1 and FOXD1,which has been verified in Sertoli cells.In this study,to explore the regulatory function of miR-2954 in the development of chicken gonads,artificially synthesized miR-2954-VMO was used to inhibit the expression of miR-2954 in chicken embryos,and an aromatase inhibitor（AI）and estradiol（E₂）was used to establish the sex reversal model of chick embryo.The effect on the expression of genes related to sexual differentiation and gonadal development provides evidence for elucidating the regulatory role of miR-2954 in the process of sex differentiation and gonadal development in chickens.The main results obtained in this study are as follows:1.By comparing the effect on the survival of chicken embryos between in vitro shell-free culture and egg windowing,the survival rate of chicken embryos in egg windowing group is higher which reached 52%after the 15th day.The result showed egg windowing is better for embryos culture in the following study.2.Successfully deliver miR-2954-VMO into chicken embryos through microinjection combined with egg windowing,the expression of miR-2954 was significantly inhibited in the gonads at days 10,12,and 18 and in the brain,muscle and gonads at days 12,establishing a feasible platform for gene function research at the chicken embryo level.3.On the 10,12,and 18th days after miR-2954-VMO injected,the morphology of gonads was processed by HE staining,and the expression of genes related to sex diferentiation were measured by RT-qPCR and immunofluorescence.The results showed that the morphological changes of chicken embryos gonads were not obvious.The miR-2954 inhibition in male embryos reduced the expression of male sex differentiation genes DMRT1,SOX9 and AMH,while the expression trend of female maker genes CYP19A1 and FOXL2 was not obvious.The protein expressions of DMRT1 and SOX9were decreased in male gonads,while CYP19A1 was slightly expressed.The results showed that miR-2954 could regulate the expression of gonadal differentiation related genes DMRT1,SOX9,and CYP19A1 in chicken embryos.4.HE staining,RT-qPCR and immunofluorescence were used to detect the morphology of embryonic gonads and the expression of genes related to sex differentiation at the 10th day after the chicken embryos were treated with aromatase inhibitor（AI）and estradiol（E₂）on day 3.The results showed that the gonads of the female embryos were symmetrically distributed,similar to the control males after AI-treated.The expression of female sex differentiation genes CYP19A1 and FOXL2 were dramatically decreased,while the expression of male sex differentiation genes DMRT1and SOX9 were significantly up-regulated;The protein expressions of CYP19A1 and FOXL2 were decreased while the protein expressions of DMRT1 and SOX9 appeared.In the male embryos treated with E₂,the degeneration of the right gonadal gland became smaller,and part of the tissue morphology was similar to that of the female ovary.The expression of male sex differentiation genes DMRT1 and SOX9 were significantly down-regulated,while the expression of female sex differentiation genes CYP19A1 and FXOL2 were up-regulated.These results indicate that the use of aromatase inhibitors and estrogen reverses sex of gonads in chicken embryos.5.RT-qPCR was used to measure the expression of miR-2954 in the gonads of chicken embryos treated with AI and E₂.The results showed that the expression of miR-2954 was significantly up-regulated in the female embryo gonads after AI-treated,but not in the male embryo gonads after E₂-treated.The results of sex reversal chicken embryo model showed that the expression of miR-2954 was consistent with the change trend of male sex differentiation gene DMRT1 and SOX9,indicating that miR-2954mainly regulates the differentiation and development process of male testis.These results indicate that miR-2954 in chicken gonads is likely to have important roles in sexual development,but the further study of miR-2954 exact regulatory status is still needed.