Study On The Potential Effect Of IGF2BP3 And 42Sp50 In Oocyte Development By Using GSDF Knockout Medaka

Germ cells have difficult ways of regulating the sex differentiation into sperm or egg cells.The germ cells of the medaka?Oryzias latipes?under the control of their own foxl3 factor and the dmy sex determinant of somatic cells enter female and male differentiation.Among them,gonadal soma derived factor?gsdf?is a direct downstream factor of dmy,its knockout can lead to XY feminization,transgene overexpression can lead to XX testis,but gsdf biological role and target genes Still not clear.XY female can be created by genetic manipulation of the gsdf,through transgenic over-expression or targeted disruption of gsdf in medaka?Oryzias latipes?,however,the bio-function and target genes of gsdf are still unclear.We used Label Free proteomics combined with MS mass spectrometry to quantitatively compare the protein expression profiles in normal XX ovary,XY testis,and gsdf-deficient XY,found that gsdf-deficient ovaries of Igf2bp3 and 42Sp50 in the ovary significant changes in expression were observed.Compared with the normal female and male gonads,Igf2bp3 and 42Sp50 proteins were highly expressed in gsdf-deficient XY ovaries,and the igf2bp3 and 42Sp50 gene transcription and translation were greater in gsdf-deficient ovaries than in normal ovaries by using real-time PCR quantification.The igf2bp3 and genomic structure located in the same region of the chromosome are highly conserved in fish and mammals.Cell positioning with anti-human H3K27me3/IGF2BP3 antibody,we found The expression of igf2bp3 is related to oocyte development.In one month old gsdf-deficient ovaries,clustered germ cell cysts increased significantly compared with normal ovaries.There were h3k27me3and igf2bp3 positive germ cell clusters in a single cyst,and both were negative Germ cell clusters,comparing of h3k27me3 and igf2bp3 co-positive germ cell clusters in normal female ovaries.In normal male testis,there are fewer igf2bp3 positive germ cells,indicating that the absence of gsdf eliminates the inhibitory effect of gsdf on X-germ cells,thereby allowing germ cells to enter igf2bp3-mediated oocyte development.Depletion of gsdf led to an abnormal increase in cystic germ cells growing in the one-month-old XY ovary,which expressed high igf2bp3 in some zygotic and thick-line stage oocytes,but igf2bp3 was silent in germ cells and other thick-line stage oocytes.Therefore,at least two oocyte populations appear after the onset of meiosis in the gsdf^-/-ovary.Our results provide new insights into the gsdf-igf2bp3 signaling pathway.These signaling mechanisms are the basis of the basic process of gameteogenesis;these mechanisms may be very conservative on evolutionary pathways.As an important RNA-binding factor,igf2bp3 can induce cell proliferation and participate in the regulation of signaling pathways in cell development.Cells that overexpress igf2bp3 show increased tumor growth.Anti Miillerian Hormone?amh?is a member of the transforming growth factor-??TGF-??superfamily.AMH treated pregnant mice showed a PCOS-like phenotype in adulthood[1].Knockout of gsdf,amh homologous proteinin,in medaka,zebrafish,and tilapia can produce primary oocyte proliferation,similar to the PCOS phenotype,in this experiment,we using gsdf^-/-XY medaka as a model study found that igf2bp3 can regulate the development of oocytes,mediate the development and differentiation of oocytes to primary oocytes.In pathological tissue sections,germ cells highly express igf2bp3,which is expected to provide a new diagnostic molecular markers for seminoma and polycystic ovary syndrome.The expression of e EF1a including 42Sp50 was examined in gonads during sexual differentiation,and mature gonads of normal ovary or testis,or gsdf depletion ovary by immunofluorescence using anti-human EEF1a antibody.Evidence of normal XX distinct from gsdf-deficient XY oocytes by dual-color immunofluorescent analysis,provides a new clue for the mechanism of vertebrate gametogenesis.In this experiment,we focused on the key factors igf2bp3 and 42Sp50 under the regulation of gsdf to explore the important regulatory role of gender differentiation,which may play an important role in the process of female differentiation.When gsdf is missing and the male differentiation signal pathway is closed,Igf2bp3 regulates germ cells to egg cells Continuing development,42Sp50 is regulated by gsdf and its expression level is increased,which can participate in maintaining the development of female germ cells.