| As an important biological process,sex determination has always been a hot issue in biology,reproduction,developmental biology and animal science.Due to the differences in productivity and economic benefits between males and females of economic animals,the study of the mechanism of sex determination and the realization of sex identification and control has also become an important issue in production.Chicken is an important economic and model animal.It can be used not only as an easy-to-obtain model animal for scientific research,but also as a source of high-quality animal protein for human beings.The sex of chicken is closely related to its production benefit.In broiler production,the production benefit of rooster is higher than that of hen,while in laying hen production,the production benefit of hen is much higher than that of rooster.Therefore,if we can obtain single-sex cocks in broiler production or single-sex hens in laying hen production through the regulation of sex,the breeding costs due to chick culling can be greatly reduced.this will not only bring huge economic benefits to the entire breeding industry,but also avoid the ethical problems caused by chick culling.Therefore,the realization of chicken sex control is a key problem to be solved in the poultry industry.It is well known that in mammals,key genes for gender determination have been excavated.The Sry gene in mice can specifically determine male development.Although some genes have been found in poultry,such as Dmrt1,Amh and Foxl2,the subsequent experiments show that these genes can only participate in the process of gender determination in poultry,which greatly limits the development and application of chicken gender control technology.Based on this,the gene expression and influencing factors of key time points in the process of chicken embryo sex determination were systematically analyzed by transcriptional sequencing technology in this study.It was found that the Hintw gene on chicken W chromosome was the key factor in the process of chicken sex determination.The biological function of the gene in gender determination was proved through in vivo and abroad experiments,and its molecular mechanism was analyzed.It provides a reference for the research of the mechanism of chicken(bird)gender determination,and provides theoretical support for further research on the regulation network of chicken gender determination.The results are as follows:(1)Transcriptome analysis of key factors in chicken sex determinationThe transcription group sequencing of ESCs and PGCs in the early stage of embryo development showed that there was a difference in the expression of sex gene in early blastocyst(ESCs),indicating that the sex decision of chicken had begun in the early embryonic development(ESCs stage).By GO analysis,energy metabolism and epigenetic modifications(DNA methylation and histone acetylation)were determined to be involved in the chicken sex determination process.Three signal pathways,such as pathway in cancer,metabolism of xenobiotics by cytochrome P450 and drug metabolism-other enzymes,were identified to play important roles in the process of chicken gender determination.The key factors in the process of chicken sex determination were determined by gene interaction,because Cyp19a1,Dmrt1,Sox9,Amh,Hintw and Wnt4 were the key factors in the process of chicken sex determination.(2)Study on the function of energy metabolism and epigenetic modification in the early sex determination of chickenThe levels of glucose uptake and lactate accumulation in female and male ESCs and CEF were examined,and it was found that glycolysis levels were higher in male ESCs and CEF and lower in female cells.The levels of HAT in ESCs and CEF were detected by ELISA.It was found that the level of HAT in male ESCs and CEF was significantly higher than that in female cells(P<0.01).The DNA methylation level of male ESCs was significantly lower than that of female ESCs(P<0.01).The function of chicken gonad was studied by adding glycolysis activator(2i:SB431542+pd0325901),histone deacetylation modifying enzyme inhibitor(VPA)and DNA methylation inhibitor(Vc).HE staining results showed that:compared with the normal development of male and female gonads,the cortex of ovaries was significantly thinner and the medulla became more denser after adding 2i,VPA or Vc.QRT-PCR results showed that compared with normal ovaries or testes,Sox9 and Amh in ovaries and testes treated with 2i,VPA or Vc were significantly up-regulated(P<0.05),while Foxl2 and Wnt4 were significantly down regulated(P<0.05).ELISA showed that 2i,VPA or Vc treatment could inhibit the level of estradiol in ovary and testis and increase the level of testosterone(P<0.01).Thus,activating glycolysis can promote the process of male sex determination,while inhibiting the level of DNA methylation modification and increasing the level of histone acetylation modification can also promote the process of male sex determination in chickens.(3)Screening of key genes for chicken sex determinationIn this study,iPS similar to ESCs were obtained through the establishment of in vitro CEF reprogramming system,and the process of IPS reprogramming was optimized by treating CEF with glycolysis activator(2i:SB431542+pd0325901),DNA methylation inhibitor(Vc)and histone deacetylase inhibitor(VPA).High quality total RNA was obtained from CEF and iPS samples.The effects of energy metabolism,DNA methylation and histone acetylation treatment on sex related genes were detected by using transcriptome sequencing(Illumina sequencing),and the differential gene expression changes in the process of sex determination were compared at the gene level.It was found that the addition of 2i activated the level of glycolysis to promote the process of male determination.while the addition of Vc reduced the level of DNA methylation in cells and the addition of VPA increased the level of histone acetylation,which could also promote the process of cell male determination.KEGG and go analysis showed that 2i and Vc/VPA regulated male sex determination genes or signaling pathways were conserved during male and female CEF reprogramming.At the same time,a Wayne analysis of candidate genes in the male and female CEF reprogramming process revealed 45 key candidate genes for 2i and Vc/VPA regulation of sex determination in chickens.Finally,the interaction analysis of key sex candidate genes in chicken ESCs showed that Hintw was the core of the regulatory network of chicken embryo sex determination process.(4)Function of Hintw in chicken sex determinationThe expression levels of Hintw in male and female cells,early hermaphroditic embryo development,gonadal development and different tissues of male and female chicks were systematically detected,andit was found that Hintw was differently expressed in both sexes,with high expression in female embryonic and gonadal development and tissues,and low expression in male embryonic and gonadal development and tissues.Sh-Hintw lentiviral interference vector and OE-Hintw lentiviral overexpression vector were successfully constructed,and both of them could significantly interfere with or enhance the expression of Hintw.Through in vivo functional verification test,it was found that overexpression of Hintw in male chick embryos could lead to asymmetric testicular development at 18.5 days.HE staining results showed that compared with normal development of male and female gonads,the cortex of male gonads overexpressing Hintw was significantly thickened,the convoluted tubules in testis collapsed,and the interior became loose,showing the morphological results of ovary.However,the cortex of female gonad which interfered with Hintw became thinner and the medulla of ovary appeared the shape of convoluted tubules.QRT-PCR results showed that overexpression of Hintw could significantly inhibit the expression of male related genes Dmrt1,Sox9 and Amh(P<0.05),and promote the expression of female related genes Cyp19a1 and Foxl2(P<0.05);interference with Hintw had the opposite effect.At the same time,overexpression of Hintw in male chick embryos can inhibit the level of testosterone and increase the level of estradiol,but interference with Hintw in female chick embryos can inhibit the level of estradiol and increase the level of testosterone.These results indicate that Hintw was involved in the sex determination process of early chick embryos,which laid a foundation for further elucidating the molecular regulatory network of sex determination process in chick embryos.(5)Molecular mechanism of Hintw in chicken sex determinationThe interaction protein UBE2I of hintw was successfully obtained by pull down and mass spectrometry.Further verification by Co-IP showed that in female cells,HINTW could bind to UBE2I,while HINTZ could not bind to UBE2I.The expression of Ube2i in chicken embryo was detected.It was found that the expression level of Ube2i in female tissue was significantly higher than that in male tissue(P<0.01),and the expression level in female ovary was significantly higher than that in other tissues.In vivo functional test by lentivirus mediated overexpression/interference of Ube2i showed that after overexpression of Ube2i,the testicular cortex of male chick embryos developed,the medulla showed loose structure,the seminiferous tubules decreased,and the female gonads showed characteristics.At the same time,the expression of Foxl2,Hintw,Cyp19a1 and other female related genes were significantly up-regulated(P<0.01),while the expression of Sox9,Dmrt1,Wt1 and other male related genes were significantly up-regulated.The content of estradiol in male chick embryo was higher than that in the control group,and the content of testosterone was lower than that in the control group,while the opposite trend was observed after disturbing the expression of Ube2i.These results indicate that Ube2i can effectively promote the differentiation of chicken embryos toward females.Taken together,this experiment successfully screened and identified Hintw as a key gene in the process of chicken female sex determination.Hintw interacts with female sex related gene(Ube2i)to participate in the early sex determination process of chicken embryo.The results of this experiment laid a theoretical foundation for further analysis of the molecular mechanism of chicken sex determination. |
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