| Poultry sex determination and gonadal development is regulate and control by the several genes and in vivo hormone levels.By far,themechanisms of poultry sex determination and regulation was still unclear.On the other hand,there were several research showed that the fadrozole can inhibits the biosynthesis of estrogen and cause the sex reversal of ZW embryos.Due to the short hatching period and low costs,the sex reversal of chicken embryos has become a hot point of avian sexual model.At present study,we focus on the Fadrozole injectionof air chamberof embryo eggs at E3.5,which would cause the sexual reversal of chicken embryos.Then we examined the polymorphism of CHDI gene to found out the heredity sexuality and observed changes of sexual gland in chicken embryos.The expression of four genes(DMRT1,OX9,FOXL2,and RSPO1)in chickens gonads of E8,E10,E14,E19,and 1wk(1 week old)were detected by RT-qPCR.Besides we also detected the expression of SOX9 and RSPO1 protein in the E19 embryonic gonads by Western-Blot.The resultswere as follow:After the treatment of Fadrozole,The ZZ Fadrozole treated group was similar to the ZZ control group,both gonads were rod-like,the size was similar,and the morphological structure was consistent with the control group.But some ZW individuals with masculinization characteristics were found,gonad of the left side in ZW masculinization individuals was rod-like,with testicular characteristics rather than flat with angular ovarian characteristics.In addition,there were some individuals on the right side of the gonads didn’t shrink atrophy,the size was close to the left gonad.Histological sections showed that the seminiferous tubules appeared in the medulla,cortical layer didn’t find the structure of the primordial follicles as in the ZW control group.In the control group,DMRT1 and SOX9 genes have higher expression in male(ZZ)gonad in various periods,whileFOXL2 and RSPO1 genes have higher expression in female(ZW)gonad,and the difference is significant between genders(P<0.05).Compared with high expression of DMRT1 and SOX9 in testes and FOXL2 and RSPO1 in ovaries,the four genes have lowerexpression in heart,kidney,liver and leg muscles,but the expression of SOX9 and FOXL2 in brain tissues is second only to the high expression of the gonad.After the treatment of Fadrozole,the difference of four genes between the control group and the treatment group in the gonad of ZZ(male)was not significant(P>0.05).However,there was significant difference between that in ZW(female)(P<0.05),suggesting that the effect of Fadrozole on female gonad was obvious.In the ZW group,the expression of DMRT1 and SOX9 was significantly higher than that of the ZW control group(P<0.05),the former decreases with time,whereas the latter gradually increases.The expression of FOXL2 and RSPOl genes was significantly lower than that of the ZW control group(P<0.05),and the growth of chick embryos was gradually increased.In the treated group,the expression of SOX9 protein in ZW chickens gonad was significantly higher than that in ZW control group by Western-Blot,but the expression of RSPOl protein in ZW chickens gonad was significantly lower than that in ZW control group.Differences of the two proteins expression in ZZ gonad between treatment group and control group is not obvious.Those results were consistent with the results of fluorescence quantification as shown above.In conclusion,DMRT1 and SOX9 genes were mainly expressed in male gonads,while FOXL2 and RSPOl genes were mainly expressed in the ovary.Male characteristics were presented by treatment with fadrozole in ZW chicken embryo gonads,and the treatment leaded to the up-regulated of DMRT1 and SOX9,while the down-regulated of FOXL2 and RSPO1.The same pattern also showed in SOX9 and RSPO1 protein. |
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