Discussion Of Non-gsdf Male Pathway In Medaka

Medaka(Oryzias sakaizumii and Oryzias latipes),different from other teleosts,has a clear XX/XY sex determination system,which is a good model for the study of sex determination and sex differentiation in teleosts,It has the advantages of embryonic transparency,life cycle of sexual maturation and small size of genome.Dmy as male determining gene on the medaka Y chromosome initiates the development of undifferentiated gonads to the testis by regulating gsdf,dmrt1 and other factors on the chromosome.At present,there are two species of medaka in the laboratory: Hd-rR(Oryzias latipes)and HNI(Oryzias sakaizumii).Although the male differentiation of gonads was determined by the male sex determinant dmy,there were significant differences in the number of germ cells and the expression of dmrt1 and gsdf during incubation,and the response mechanisms of the two lines were different after androgen treatment.These results suggest that there may be different signals regulating male differentiation of germ cells.The gsdf(gonadal soma-derived factor)gene is a member of the TGF-? superfamily of growth transforming factors.The transforming growth factor superfamily consists of a large number of structure-related factors,including TGF-?,bone morphogenetic protein(BMP),AMH,activin and inhibin.TGF-? signal transduction controls a variety of cellular processes,such as cell proliferation,recognition,differentiation,apoptosis,and development.From flies and worms to mammals,they play a vital role during embryogenesis and during tissue maturation.Studies have shown that gsdf exists in teleosts,and is very conserved in teleosts.As a downstream gene of dmy,male gonadal differentiation is regulated.It was found in the laboratory that different species of medaka responded differently to gsdf deletion.In Hd-rR strain,the deletion of gsdf resulted in the feminization of XY-type medaka,and the mutant had large abdomen and large ovary,and there was only a thin layer of germ cells.Most of the oocytes were only primary oocytes,and it was difficult to ovulate after the ovary matured,and the oocyte spawning rate and fertilization rate were very low.In HNI strain,the deletion of gsdf did not lead to the feminization of XY-type medaka,but the mutant still had large abdomen,large size of testis,The structure of some testis was disordered from the distal to the proximal part,and there was no distinct structural differentiation from the distal to the proximal part.Increased in germ cells.According to this interesting phenomenon,we took one-month-old wild-type testis RNA of wild-type Hd-rR strain and one-month-old wild-type ovary RNA and one-month-old knockout giant ovary RNA and one-month-old knockout giant testis RNA of HNI strain,respectively.The expression of RNA-binding protein Igf2bp3 in knockout ovary was much higher than that in wild-type ovary,and the expression level was 300 times higher than that in wild-type ovary,on the contrary,there was little expression in testis.Far lower than the wild-type ovary.At present,most of the research on igf2bp3 is in mammalian cancer and the study on the growth,development and metabolism of organisms.Study was still rare in fish.In this study,the igf2bp3 gene was cloned for the first time,and the RT-PCR,QPCR,two-color immunohistochemical double color and fluorescence in situ hybridization techniques to exploring the igf2bp3 in medaka mRNA expression and subcellular localization of gonad and embryos.The results showed that igf2bp3 mRNA was expressed in medaka brain,liver,ovary and testis,with the highest expression in ovary,and in all stages of embryonic development.Igf2bp3 protein was highly expressed in knockout ovary,mainly expressed in oocytes at meiosis stage,are rarely expressed in knockout testis,suggesting that igf2bp3 gene mediates ovarian development.Yeast two-hybrid screening showed that TGF-? receptor amhr II binding to gsdf,through in situ hybridization of gsdf and amhr II,it was found that there was no difference in the expression of amhr II in the knockout giant testis compared with the wild type testis.So we hypothesized that the presence of giant testis in the HNI strain was not regulated by the gsdf-amhr II male pathway.The results of in situ hybridization between gsdf and dmrt1 showed that there was no difference in the expression of dmrt1 between knockout giant testis and wild type testis,suggesting that the appearance of giant testis in HNI strain was not regulated by gsdf-dmrt1 signaling pathway.Through the comparative study of two species of medaka,we found that there is a non-gsdf male pathway in medaka.We have discovered igf2bp3 factor,which plays an important role in the development of oocyte.Oocyte development is conservative in lower vertebrates.The functional analysis of Igf2bp3 will provide a theoretical basis for further understanding the mechanism of oogenesis and spermatogenesis in teleosts.These mechanisms may provide a good reference for the study of molecular mechanisms and roles in vertebrate reproductive development.