Investigation Of Changes In The Chromatin Accessibility And Transcriptome During Early Development Of Geese Ovaries

The reproductive performance of poultry,especially the laying rate,depends on the development of ovary and follicles,and the early development of ovary directly affects the final laying ability.The laying performance is closely related to the key events of early ovarian development,such as the dramatic changes in the number of oocytes before and after hatching,primordial follicle formation and its initiation and recruitment.So far,however,there were few studies on poultry,especially on geese with poor laying performance,which was almost blank.This experiment was based on the observation of ovarian histomorphology of geese at different stages of E6,E12,E15,E26,P0,P4,P7,P14,P21 and P28,using RNA-seq and ATAC-seq technology,bioinformatics analysis was conducted on the follicular stage of germ cells(E15),the newborn stage(P0),the primordial follicular formation stage(P4),and the primary and secondary follicular formation stage(P28),aimed at analyze the open changes of transcriptome and chromatin,screen out the related genes and transcription factors during the early development of goose ovary,The main results were listed as follows:(1)HE staining geese left ovary showed that the ovary at stage E15 was composed of germ cell cyst,the oocyte was visible in the cyst,the oocyte volume increased in the P0 stage,and the cyst is reptured,the oocyte forms a primordial follicle with the surrounding somatic cells.At the P28 stage,secondary follicles were observed.(2)RNA-seq was applied to E15,P0,P4 and P28,analysis of differentially expressed genes showed that the number of differentially expressed genes decreased first and then increased with the increase of embryo age or day age.The largest number of differentially expressed genes was found in P0 VS E15,suggesting that complex biological processes may exist in this stage.In P0 VS E15 and P4 VS P0 stages,the number of up-regulated differential genes was more than the number of down-regulated genes,while in P28 VS P4 stages,the number of down-regulated genes was more than the number of up-regulated genes.The results of GO and KEGG analysis indicated that the differential genes in P0 VS E15 stage were significantly enriched in biological processes such as translation,DNA repair,chromosome segregation,mitotic chromosome condensation,cellular protein metabolic process,gluconeogenesis.Differential genes involved in cell cycle,DNA repair,mismatch repair and other signaling pathways were down-regulated,while differential genes in biological processes such as negative regulation of angiogenesis,multicellular organism growth,and extracellular matrix organization were up-regulated,and participate in MAPK signaling pathway,ECM-receptor interaction,adipocytokine signaling pathway were up-regulated,indicating that these differentially expressed genes may be involved in the regulation of ovarian cell proliferation,differentiation and apoptosis.P4 VS P0 stage the difference of gene significantly enriched in biological processes of mitosis,protein metabolism etc,participate in regulation of protein catabolic process,Fox O signaling pathway,p53 signaling pathway and so on are up-regulated,and participate in the MAPK signaling pathways,alpha-Linolenic acid metabolism are down-regulated,suggests that these differences in gene may be related to the process of apoptosis and oocyte formation of primordial follicles.The differential genes in P28 VS P4 stage are significantly enriched in long-chain fatty acid import and metabolic process,fatty acid catabolic process,regulation of lipid metabolic process and other biological processes,The differential genes involved in SNARE interactions in vesicular transport,Wnt signaling pathway,fatty acid biosynthesis,p53 signaling pathway and other glycan degradation was up-regulated,indicating that these differentially expressed genes may be involved in the initiation recruitment of primordial follicles.(3)Using atac-seq technique,the ovaries of geese at E15,P0,P4 and P28 stages were sequenced with open regions of differential chromatin.The results showed that with the increase of embryonic age or age,the number of chromatin intervals that signal enhancement and attenuation gradually decreased.In P0 VS E15,the number of chromatin intervals with signal attenuation was higher than that of signal-enhanced chromatin interval.In P4 VS P0 and P28 VS P4,the number of chromatin intervals enhanced by signal is greater than the number of chromatin intervals with weakened signals.Further,the differential chromatin open region was mapped on the whole genome,and it was found that the P0 VS E15,P28 VS P4 signal enhanced peaks were in the intron region,and the weakened peaks were in the distal region;The result of P4 VS P0 is the opposite.Mortif predictive analysis of differential chromatin open regions showed that at the P0 VS E15 stage,the predicted transcription factors were HNF1 b,NF1,Fosl2,Hnf1,Jun B,Nr5a2,Esrrb,TEAD4,EAR2 TEAD2,In P4 VS P0 stage,the predicted transcription factors are SF1,Esrrb,Nr5a2,TGA6,HOXA1,Lhx2,Lhx1,Lhx3,Nkx6.1,Dlx3.In the P28 VS P4,the predicted transcription factors are GATA3,NF1-halfsite,GATA1,GATA2,GATA6,HNF1 b,Hnf1,ATHB13,ATHB20,ATHB5.(4)Combined analysis of ATAC-seq and RNA-seq showed that changes in the chromatin open region signal of P0 VS E15,P4 VS P0,P28 VS P4 promote or inhibit the change of gene expressions.The analysis of the relationship between differential gene and transcription factor targeting regulation indicates that the chromatin open state is consistent with the trend of gene transcription level and the gene expression level was up-regulated or down-regulated by the top 5 transcription factor-encoding gene pairs.In stage P0 VS E15,they were BATF-MRAP 、 NF1-ZBTB16 、 NF1-halfsite-ZBTB16 、 NF1-ZBTB16 、NF1-halfsite-ZBTB1,TEAD4-VGLL2 、 TEAD4-CALB1 、 Esrrb-LOC106038206 、Tcf21-FAM19A2 、 NF1-SHISA3,respectively.In stage P4 VS P0,they were Lhx2-LOC106045017、ATHB25-HOXD4、Lhx1-PCK1、Nkx6.1-SPP1、Lhx2-SLC13A1,HOXA1-LRRC4C、TGA6-PCDH15、SF1-PRKCB、TGA6-UBXN4、LIN-39-ZNF451,respectively.In stage P28 VS P4,they are At5g05790-LOC106045491、GATA3-SCEL、GATA3-G0S2 、 GATA3-CACYBP 、 NF1-halfsite-CALB1,ATHB13-DPYS 、Hnf1-SLC22A7、HAT2-HOXD9、ATHB6-AHSG、HNF1b-LOC106036766,respectively.In summary,using ATAC-seq and RNA-seq preliminary reveal that differentially expressed genes enriched in GO,KEGG,chromatin openness changes,targeted regulatory relationship between transcription factors and differentially expressed genes during early development of goose ovary.thus laying a theoretical foundation for mapping the core transcriptional regulatory network during the early development of goose ovary.