The Expression And Mechanism Of RSPO1 In The Development Of Chicken Embryo Female Gonads

In nature,most animals have sexual reproduction,which is dependent on the female and male gonads.The gonad is the only organ in the animal that undergoes two distinct developmental processes in different sexes,developing into a testis and producing sperm in males and developing into an ovary and producing eggs in females.The gonads before the early sex differentiation of chicken embryos have the same phenotype and structure between male and female,which is called bipotential gonads.After the beginning of sex differentiation,the male gonad cortex degenerated and the medulla developed into testis.The female gonadal cortex continues to develop and thicken,developing into an ovary.In this process,some genes and signaling pathways related to sex determination and gonadal differentiation play an important regulatory role.DMRT1 is recognized as a determinant of male testicular development,but the molecular mechanism of female ovarian development is not very clear.Studies on vertebrates have shown that RSPO1 is involved in the ovarian development of many species through the RSPO1/WNT4/β-catenin pathway.In view of this,this study first sequenced the chicken embryo RSPO1 gene and detected the temporal and spatial expression patterns in chicken embryo tissues.Then,the sex reversal model was constructed by injecting FAD(aromatase inhibitor)and E2(estrogen),and the gonadal cells were treated with E2 to study the effect of estrogen level on the expression of RSPO1 and related pathway genes.Finally,we explored its mechanism by overexpressing RSPO1 in male gonad cells.The main results of this study are as follows:(1)RSPO1 gene analysis and temporal and spatial expression in chicken embryo tissues.By comparing the amino acid sequence of RSPO1 protein in chicken and other species,it was found that the similarity between species was high,which indicated that RSPO1 may have similar functions in ovarian development of chicken and other species.By detecting the expression of RSPO1 in different tissues of E12 chicken embryos and gonads at different stages of incubation,it was found that the expression of RSPO1 in gonads and spleen was the highest in all tissues of E12 chicken embryos,and the expression of RSPO1 in female gonads was significantly higher than that in males.WB results showed that the expression level of RSPO1 protein in female gonad of E12 chicken embryo was significantly higher than that in male gonad.The localization of RSPO1 protein by immunofluorescence showed that it was mainly expressed in the cortical layer of gonads,and the expression level in female gonads was significantly higher than that in male gonads.It can be seen that RSPO1 showed female-biased expression in chicken embryo gonads.(2)Effects of estrogen levels on RSPO1 expression in chicken embryonic gonads.In the FAD(E2.5 day treatment)-induced male E12 female gonads,compared with the control group,RSPO1 mRNA and protein expression levels were significantly down-regulated,female gonad development-related genes FOXL2 and CYP19A1 were significantly down-regulated,and male gonad development-related genes DMRT1 and AMH were highly and significantly up-regulated,respectively.In E2(E2.5 days treatment)-induced feminized E10.5 male gonads,the mRNA expression of RSPO1 was significantly up-regulated,FOXL2 and CYP19A1 were significantly up-regulated,and DMRT1 and AMH were significantly down-regulated.In the isolated and cultured gonad cells,there was no significant difference in the mRNA expression of RSPO1 between female and male(both low).However,after male gonad cells were treated with different concentrations of E2 and female gonad cells were treated with medium concentration of E2,the expression of RSPO1 was significantly up-regulated,and the expression of female gonad development-related genes CYP19A1,ERa and β-catenin in the medium concentration treatment group was significantly up-regulated.It can be seen that there is no inherent difference in the expression of RSPO1 in gonad cells between genders,and its expression is induced by estrogen,that is,the female-biased expression of RSPO1 in chicken embryo gonads is E2(estrogen)dependent.(3)The mechanism of RSPO1 gene in gonadal cells.By overexpressing RSPO1 in male gonad cells and performing transcriptome sequencing,the genes that may play a role were screened.A total of 182 differentially expressed genes were screened by transcriptome data analysis,including 128 up-regulated genes and 54 down-regulated genes.PTGS2,CORO2A,ECM1,ISYNA1,CPED1 and other genes related to ovarian or testicular development were verified by qPCR.In addition,the expression of ovarian development-related genes was detected after overexpression of RSPO1.It was found that the downstream gene WNT4 of RSPO1 was significantly up-regulated,and the downstream gene ERa of E2 pathway was significantly up-regulated,but the upstream genes FOXL2 and CYP19A1 of E2 pathway did not change significantly.After further overexpression of WNT4 and RSPO1 in male gonadal cells,the E2 pathway genes FOXL2,CYP19A1 and ERa were significantly up-regulated.The above results indicate that RSPO1 can act on genes related to ovarian or testicular development,and may synergistically promote the expression of E2 pathway genes through the downstream gene WNT4 to promote ovarian development.In summary,RSPO1 shows a non-cell-inherent,estrogen-dependent female-biased expression in chicken embryonic gonads,and synergistically promotes female ovarian development by acting on downstream related genes such as WNT4.