| The study of gonadal function is of great theoretical and practical importance because gonadal development is a complex process involving sex determination,differentiation and maturation as testes or ovaries,and the level of gonadal development in male and female geese also determines their performance.In mammals,gonads develop symmetrically on the right and left sides,while in poultry they develop asymmetrically,with males developing symmetrical testes on the right and left sides and females not developing on the right side,Asymmetrical gonad development in poultry has been shown to be regulated by several signalling pathways,of which the TGF-β/Smad and MAPK signalling pathways are the main pathways affecting gonad development.The TGF-β/Smad and MAPK signalling pathways are the main signalling pathways affecting gonadal development.Therefore,the present study investigated the morphological and structural features of the developing goose gonads and the expression and regulation of both signalling pathways during their development,laying the foundation for further research into the regulatory mechanisms of goose gonad growth and development.In this study,the embryonic gonads of Jilin white geese were collected from day9 to day 28 of embryonic development,and the morphological changes were observed through a body microscope,and the genomic DNA of the leg muscle tissue was extracted to identify the sex of the embryos using PCR amplification of the CHD1 gene.In addition,the expression and distribution of TGF-β/Smad and MAPK signalling pathway genes in male and female gonads were analysed during development.Finally,the TGF-β/Smad and MAPK signalling pathway activators TMAO and TBHQ were injected into poultry embryos to detect the localisation and expression levels of the main genes and proteins of each signalling pathway in the male and female gonads,with a view to exploring the status of the goose embryonic gonads and their changes in a more in-depth and systematic manner:(1)Histological observation of the different modes of gonad development in the goose embryo.The results showed that under the microscope,the embryonic gonads of the goose developed on both the left and right sides,with the male gonads developing symmetrically,with the left side slightly larger,and the female gonads developing asymmetrically,with the left side developing and the right side degenerating.The female gonads grow immediately adjacent to the mesonephros and are clearly distinguishable from the cortex and medulla.The medulla of the female gonads was observed to contain a large number of vacuolated structures on embryonic day 12(E12),and combined ALP-PAS staining showed that the primordial germ cell lines of geese differentiated at different times,with male gonads fully differentiated on embryonic day 18(E18)and female gonads fully differentiated on embryonic day19(E19).(2)Study on the expression pattern of TGF-β/Smad and MAPK signalling pathways during the development of goose embryonic gonads.q RT-PCR analysis showed that the TGF-β/Smad(TGF-βRⅠ,TGF-βRⅡ,Smad2,Smad3)and MAPK(MAPK1,MAP2K1,MAP2K2)signalling pathways in the gonads of goose embryos from E9 to E28 at embryonic stage,MAP2K2 signalling pathways,with high expression in E14,E18 and E23.The expression of TGF-β/Smad downstream proteins Smad3 and Smad2 was specifically detected by Western Blot,and it was found that the relative expression level of Smad3 protein in E28 was highly significant compared with other embryonic ages(P < 0.01),and the expression in male gonads was higher than that in females(P < 0.01);the expression of Smad2 protein in E17 and E19 was highly The expression of Smad2 protein was highly significant in E17 and E19(P <0.01)and higher in E28 female gonads than in males(P < 0.01).In the MAPK signalling pathway,the expression of ERK1/2 proteins was highly significant at E17 compared to other embryonic ages(P < 0.01)and was higher in the gonads of female embryos than in males(P < 0.001).The results suggest that TGF-β/Smad and MAPK signaling pathways are all involved in the regulation of gonad development in goose embryos to some extent.(3)Effect of embryo injection of MAPK signaling pathway activator TBHQ on gonad development in goose embryos.One hundred and sixty Jilin white goose breeding eggs were divided into four groups for embryo injection at E7: control group and 5mg/kg,20mg/kg and 40mg/kg activator injection groups.The results of q RT-PCR showed that the expression of MAPK1,MAP2K1 and MAP2K2 m RNAs in females was proportional to the concentration of TBHQ,whereas in males,the expression of MAPK1 and MAP2K2 m RNAs increased and then decreased(P < 0.05).The expression levels of ERK1/2 and Smad3 showed an overall significant downward trend(P < 0.05)at E18,while Smad2 and Smad1/5/9 showed the opposite expression trend;at E19,ERK1/2,Smad2 and Smad1/5/9 all showed a significant upward trend(P < 0.05)and Smad3 showed a significant decrease(P < 0.05).The results indicated that TBHQ 40mg/kg had the greatest effect on the gonadal growth and development of goose embryos in the MAPK signaling pathway regulation.(4)Effect of embryo injection of TMAO,an activator of TGF-β/Smad signalling pathway,on gonadal development in goose embryos.Goose embryos were injected with 50mg/kg of TMAO on embryonic day 7(E7)and the expression of key genes and proteins of the TGF-β/Smad signalling pathway was analysed at E18 and E19.The results of q RT-PCR showed that the relative m RNA expression of TGF-βRI/II genes of the TGF-β/Smad signalling pathway was significantly decreased in both male and female gonads compared to the control group(P < 0.05).(The relative m RNA expression of TGF-βRI/II genes in the TGF-β/Smad signaling pathway was significantly decreased in both male and female gonads compared with the control group(P < 0.05),while the expression of Smad2 and Smad3 were significantly increased compared with the control group(P < 0.05).The western blot analysis showed that the expression level of Smad2 was lower in the test group than in the control group at E18(P < 0.05),while it was higher in the control and injection groups at E19(P < 0.05);the expression of ERK1/2 、 Smad3 was significantly increased in the test group compared to the control group at E18 and E19(P < 0.05).The results indicated that the TGF-β/Smad signaling pathway affected the development of gonads in goose embryos,and inhibited their growth in the early stage of gonad differentiation and promoted their development after differentiation.In conclusion,the aim of this study was to investigate the basic morphology of gonad development in goose embryos,to determine the time point of gonad differentiation in goose embryos,and to determine the expression patterns of the main genes and proteins in the TGF-β/Smad and MAPK signalling pathways and their pathways after embryo injection of pathway activator,which initially demonstrated the basic characteristics of gonad development in goose embryos and the synergistic effect of the above two pathways on the regulation of gonad development in goose embryos.The results showed that the gonadal development of goose embryos was regulated by the two pathways. |
PDF Full Text Request