Study On The Function And Regulatory Mechanism Of Transcription Factor Sox3 In Oogenesis Of Nile Tilapia(Oreochromis Niloticus)

Animal oogenesis is a biological process in which female produce mature eggs.Sexually reproductive female and male individual mate to produce fertilized eggs,followed by embryogenesis.Most of the cytoplasm of fertilized eggs comes from the egg,which contains many important components that support early embryogenesis.Therefore,the quality of the egg is very important for embryonic development.Abnormal oogenesis often leads to female infertility.Therefore,it is necessary to study the regulation mechanism of oogenesis.Sox3 is a member of the Sox gene family.Studies in mice have shown that Sox3 is essential for oocyte development in female mice,and testicular differentiation and spermatogenesis in male mice.Zebrafish Sox3 is expressed in somatic cells such as granulosa cells and theca cells of the ovary,and it plays a role in zebrafish oogenesis.Our previous transcriptome analysis showed that Sox3 was highly expressed in the ovary of Nile tilapia(Oreochromis niloticus).However,the expression pattern of Sox3 in the ovary of Nile tilapia,its function and molecular mechanism in ovarian development and oogenesis remain to be studied.In this study,we used transcriptome data analysis,real-time PCR experimental verification and fluorescence in situ hybridization to explore the temporal expression and cell localization of Sox3 in Nile tilapia gonads.We also prepared anti-Sox3 antibody of Nile tilapia,and used the antibody to detect the cellular localization of Sox3 protein in the ovary by immunofluorescence assay.Then,we successfully constructed the Sox3 homozygous mutant line of Nile tilapia by CRISPR/cas9 gene editing technology.The effects of Sox3 deletion on ovarian development and oogenesis of Nile tilapia were further studied by morphological,histological analysis and lipid staining experiments.We also explored the regulatory mechanism of Sox3 during ovarian development and oogenesis by transcriptome sequencing and ChIP-seq.The main results of this study are as follows.1.The expression of in Nile tilapia,preparation and detection of anti-Sox3 antibody(1)The expression pattern and cellular localization of Sox3 in the gonads of Nile tilapia at different developmental stageIn this study,the expression pattern of Sox3 in the gonads of Nile tilapia at different developmental stages were investigated by transcriptome data analysis and Real-time PCR verification.The results showed that the expression of Sox3 in female gonads began to increase at 30 days after hatching(dah).With the development of gonads,the expression of Sox3 further increased at 90 dah,and reached the highest level at 180 dah.In males,Sox3 was only expressed in the gonads of males at 30 dah,but almost not in other stages.We analyzed the expression of Sox3 in the gonads of Nile tilapia(Oreochromis niloticus)at different developmental stages by fluorescence in situ hybridization(FISH).The results showed that Sox3 was expressed in the oocytes of phase I and phase II of the Nile tilapia ovary at 90 dah,and in the oocytes of phase I,phase II and phase III of the Nile tilapia ovary at 180 dah.(2)The preparation and detection of anti-Sox3 antibody in Nile tilapiaIn this study,Sox3 protein was induced and expressed in E.coli,and the expressed protein was purified.Finally,anti-Sox3 antibody was prepared.Western blot analysis showed that the antibody could recognize the Sox3 protein of Nile tilapia.Then we used anti-Sox3 antibody to perform immunofluorescence experiments on Nile tilapia ovary sections.The result is consistent with the result of fluorescence in situ hybridization.The results showed that Sox3 protein was expressed in phase I and II oocytes of the Nile tilapia ovary at 90 dah and in phase I,II and III oocytes of in the Nile tilapia ovary at180 dah.The above results suggest that Sox3 may play a role in ovarian germ cells and affect oogenesis.2.The function of Sox3 in ovarian development and oogenesis of Nile tilapiaWe constructed Sox3 homozygous mutant line of Nile tilapia by CRISPR/Cas9 technology.Then,the ovary of Nile tilapia with Sox3 homozygous mutation at different developmental stages was observed by morphology and histology;and the gonadal maturation index(GSI)was analyzed and the number of different types of germ cells was counted.The results of the study showed that there was no significant difference in the anatomy and histology of Sox3+/+,Sox3+/-and Sox3-/-ovaries at 90 dah and 120 dah.At 180 dah,the ovaries of Sox3+/+,Sox3+/-and Sox3-/-females began to show significant differences at anatomical and histological levels.Compared with Sox3+/+females,the GSI of Sox3+/-and Sox3-/-females decreased significantly,and the number of oocytes at stageⅢ(oocytes undergoing vitellogenesis)and IV(oocytes filled with yolk granules)decreased significantly.The accumulation of lipid droplets in the ovary was observed by BODIPY staining.The statistics of BODIPY positive cells showed that the number of oocytes accumulated with neutral lipids in Sox3-/-female ovary was significantly reduced compared with that in Sox3+/+ female ovary.These results suggest that Sox3 may be involved in the regulation of Nile tilapia oogenesis.3.The mechanism of Sox3 in the oogenesis of Nile tilapia(1)The changes of transcriptome in the ovary induced by Sox3 homozygous mutationBased on transcriptome sequencing and data analysis,this study revealed the effect of Sox3 deletion on the ovarian transcriptome of Nile tilapia at 180 dah.Collected the ovaries from 3 wild-type fish and 3 Sox3 homozygous mutant fish,respectively,and extract total RNA to perform the transcriptome sequencing.The results showed that 4000 genes were up-regulated and 3057 genes were down-regulated in Sox3 homozygous mutant females compared with the control.KEGG enrichment analysis showed that the differentially expressed genes were enriched in Fatty acid metabolism pathway,steroid biosynthesis pathway and PPAR signaling pathway.Further analysis of differentially expressed genes showed that Sox3 deletion significantly changed the expression of some genes related to vitellogenesis and lipid accumulation in the oocytes.The expression of Fshr and Ctsd,which are related to vitellogenesis,were significantly down-regulated.The expression of the Vldlr related to lipid accumulation was significantly up-regulated;the expression of the genes related to lipid accumulation,Lpl,Lpl2,Apoa4a,Apoa4b,Apolipoprotein AI,Apolipoprotein A-IV,Apoc2,Apocl,Apoeb,Fabp3,Fabp4a,and Fabp7a,were significantly down-regulated.These results suggest that Sox3 deletion can cause the expression change of some genes related to vitellogenesis and fat accumulation in the ovaries of Nile tilapia,which leads to the decrease of vitellogenesis and inhibition of lipid accumulation in ovaries.(2)The effect of Sox3 homozygous mutation on serum E2 and 11-KT levels of female Nile tilapiaBy measuring the levels of estrogen and androgen in the serum,we found that compared with Sox3+/+female fish at 180 dah,the serum E2 and 11-KT levels of Sox3+/+female fish were significantly reduced.Further study showed that the mRNA expression level of the estrogen synthesis rate-limiting enzyme gene Cyp19a1a was significantly down-regulated,and the mRNA expression level of Foxl2,which plays a positive regulatory role in the transcription of Cyp19ala,was also significantly down-regulated.These results indicate that the deletion of Sox3 in Nile tilapia leads to the decrease of estrogen and androgen levels in the serum of female fish,which may further affect the vitellogenesis and lipid accumulation of ovarian oocytes.(3)The potential target genes of Sox3 were screened by ChIP-seqIn this study,the potential target genes of Sox3 were screened and identified at the whole genome level by ChIP-seq analysis of ovarian tissue of XX female fish at 90 dah.Using MACS2 software to complete the peak detection analysis,we found that there were 12547 Sox3-enriched peaks in the ChIP-seq data.Use ChIPseeker software to calculate the peak distribution in each functional area.The results showed that 4100 peaks were located in the intergenic region,3463 peaks were located in the intron region,and 2914 peaks were located in the promoter region.MEME software analysis of the measured ChIP-seq data showed that the binding motifs of Sox3 potential target genes in Nile tilapia ovaries are mainly C(T/A/G)C(G/T/A)A(C/T/G)G(T/A/C)G(T/A)C(G/A/T)C(T/G/A)A(T/C/G).Through analysis,we found that the potential target genes of Sox3 are Vasa and Vldlr,etc.,indicating that Sox3 may be able to regulate the expression of Vasa and Vldlr to affect the development of ovarian germ cells and lipid accumulation.Of course,further studies are needed to confirm the hypothesis.The GO functional analysis of Sox3 potential target genes showed that these genes were involved in female gametogenesis,fatty acid metabolism,lipid biosynthesis,steroid biosynthesis and other biological processes.In conclusion,Sox3 is mainly expressed in the oocytes at phase I,II,and III of the ovary of female Nile tilapia.Sox3 homozygous mutation could reduce the number of oocytes in phase Ⅲ and Ⅳ,decrease the vitellogenesis and block the lipid accumulation of oocytes.The loss of Sox3 in Nile tilapia leads to the decrease of serum estrogen and androgen levels in female fish,which may affect the vitellogenesis and lipid accumulation of ovarian oocytes.In addition,Sox3 may be able to directly regulate the expression of Vasa to affect the development of ovarian germ cells,or regulate lipid accumulation in the ovary by regulating the expression of lipid accumulation-related genes Vldlr.This study is helpful to enrich and improve the molecular regulation mechanism of oogenesis in tilapia and other animals.