| As a typical model for studying sexual differentiation of teleost fish,medaka has the following characteristics: 1.The sex determination system of medaka is XX/XY,and in teleosts,it is one of the earliest species determined by sex-determining genes,dmy is a promoter of the sex determining gene of male differentiation.2.medaka can be artificially induced to reversal by exogenous hormone treatment,causing secondary sexual changes.3.Sexual maturity in three months,it has a large number of interspecific hybrid lines and inbred lines.The dmy gene can up-regulate the male pathway-related genes gsdf,dmrt1,sox9a2 and so on.And inhibit the female sex differentiation pathway.Gsdf is conserved in the teleost fish and is an important switching gene of the male differentiation pathway.During the male differentiation pathway,the gsdf gene is expressed in the embryo 36 stage,and cyp19b is expressed in the embryo 39 stage.It is known that the brain of the teleost fish is even Adulthood also exhibits significant gender plasticity.This is due to the unique gender differentiation process of the bony fish.In the study of medaka,the female expression of Cyp19b in adulthood was higher than that in males.There was no significant difference in puberty,and gender differences gradually occurred during the gonadal development.Cyp19b plays an important role in the process of cerebral differentiation,but its mechanism of action is still unclear.LH,luteinizing hormone,is an important gonadotropin,secreted by the pituitary secretory cells,acting on the gonads to bind to the membrane receptors on the surface,promoting the secretion of sex hormones,thereby promoting the development of germ cells and the maturation of the gonads.From this we have questioned whether there is an interaction between gsdf and cyp19b and lh.We first verified the protein expression of Cyp19b in wild-type male and female brains and gonads by Western blot.It was found that Cyp19b was abundantly expressed in the brain but less in the gonads with a molecular weight of 47 kda.Then,the Cyp19b in wild-type medaka and gonads was initially mapped by monochromatic immunohistochemistry.Cyp19b is mainly expressed in the pituitary gland in the pituitary gland,expressed in the testis epithelial cells in the testis,and expressed in the ovary epithelial cells in the ovary.After the Cyp19b and lh two-color fluorescence co-localization of the pituitary and gonads of wild-type medaka,it was found that there was no obvious difference in cyp19b in the pituitary during early stage of gonadal development(1 month old),and it was significantly higher that female than male in the pituitary at 2 months and later.Then,gsdf was knocked out that XY-type Sexual reversal into female was used as background to explore the potential relationship between gsdf and cyp19b and lh.The study found that gsdf knockout type medaka had no significant difference in the expression of Cyp19b and Lh signals in the pituitary gland between the young fish stage(1 month old)and the wild type male and female medaka.Cyp19b and Lh were significantly down-regulated during the adult stage,and the Lh signal was higher than the wild-type male than the wild-type female.At the same time,colocalization of Cyp19b and Lh was performed on wild-type male and female gonads and gsdf KOXY-type indigo ovary.The gsdf knockout type XY ? medaka ovary constructed has a huge ovary and highly infertile.Its ovary contains only immature oocytes,and only a small part of homozygotes is low pregnancy.Inducing gonadal development to the female pathway suggests that knockout of gsdf affects gonadal development.The results of gonad Immunohistochemistry showed that the wild-type female ovary epithelial cells was thicker,the Cyp19b expression was more,the knockout type barley was thinner,and the expression was less,and a large number of oocytes staying in the pre-yolk division stage were observed,and with the period When the expression is gradually increased,Cyp19b is localized to the granulosa cells of the inner and outer membranes.The wild type testis Cyp19b was localized in the epithelial cells of the vas deferens and was localized in the interstitial cells.At the same time,the laboratory constructed a gsdf overexpression system,which was injected into the cell stage of the fertilized egg by microinjection,and the male XX was obtained.The knockout of gsdf and transgene overexpression doubled its important role in initiating male differentiation.In the co-localization of Cyp19b and Gsdf overexpressing the pituitary gland and gonad of XX ?,the positive cells co-expressed by cyp19b-gsdf were found in both the pituitary and gonads,indicating the correlation of gsdf through the pituitary gland axis.To further determine the molecular mechanism by which gsdf regulates differentiation through the pituitary gland axis,we used RNA from wild-type and mutant gonads to obtain cDNA by reverse transcription,and further evaluated the expression of cyp19b and lhr using real-time quantitative PCR.Furthermore,the levels of estrogen and androgen in the blood of wild-type and mutant barley were determined by Plasma estradiol(E2)and testosterone(T)levels were measured with an E2 and T enzyme immunoassay kit(Cayman Chemical Company,MI,USA).It was found that gsdf knockout female cyanosis cyp19b and lhr were inhibited compared with normal females,and gsdf was overexpressed that up-regulation of cyp19b and lhr expression.Serum level of E2 and T was lower in Gsdf knockout XY? compared with normal females,gsdf overexpression XX ? androgen content increased significantly,indicating that gsdf expression stimulated androgen production.In conclusion,gsdf is required for ovarian or testis development through pituitary-gonadal axis differentiation and gonadal development,as well as expression of Cyp19b expression and gonadotropin Fsh and Lh signaling receptors.Gsdf also directly or indirectly regulates the expression of Fshr and Lhr in the gonads,thereby affecting the expression of sex hormones in the gonads,which are then fed back to the synthesis and secretion of Lh and Cyp19b in the pituitary. |
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