| Follicular growth and atresia are complex and well-coordinated physiological processes that are not only regulated by the hypothalamic-pituitary-gonad axis(HPG axis),but also by the follicular endocrine / autocrine factors.In order to further reveal the molecular mechanism of follicular development and to screen the key genes regulating follicular development,we used the high-throughput IlluminaHiSeq sequencing platform to detect the ovarian follicles(diameter <2mm,2-5mm,5-8mm,> 8mm)transcriptome sequencing,the quality of the data were compared with the reference genome,screening for differentially expressed genes and trend analysis.The GO category and KEGG annotation of the trend genes were used to find the biological function enrichment and specific metabolic pathways of the trend genes.The alternative splicing events and new transcripts in the process of bovine follicle gene expression were analyzed.Real-time quantitative PCR was carried out on some differentially expressed genes.SIRT1 and INHBA genes were further selected for functional verification.The main research contents and results of this study are as follows:1.Sequencing data statistics and analysis After four groups of samples were sequenced and filtered,the sequences with Quality scores greater than 20 and mapping accuracy greater than 99% could be used for subsequent analysis.Four groups of samples(in terms of follicular diameter from small to large,denoted by BF1-4,respectively)were aligned to the reference sequence The contrast rates were 90.0%,89.0%,90.0% and 92.5% respectively.2.Differentially expressed genes screeningPairwise comparison of different groups was carried out to screen the differentially expressed genes for trend analysis.1576 differentially expressed genes were screened in BF1-VS-BF2 group,of which 861 were up-regulated and 714 were down-regulated;1451differentially expressed genes were screened out in BF1-VS-BF3 group,In the group of B1VS-BF4,2359 differentially expressed genes were screened out,of which 1059 were up-regulated and 1,300 were down-regulated;in the BF2-VS-BF3 group,1447 differentially expressed genes were screened out,479 of which were up-regulated and 968 were down-regulated;in BF2-VS-BF4 group,a total of 1122 differentially expressed genes were screened,of which 445 were up-regulated and 677 were down-regulated;a total of 2403 differentially expressed genes were screened out in BF3-VS-BF4 group,of which 1310 were up-regulated and 1093 were down-regulated.3.Differentially expressed genes trends and functional enrichment analysisThe collection of significant difference genes were selected as the research objects to analyze the trend,and 8 significant change trend models(P<0.05)were obtained.The up-regulated genes and the down-regulated genes were combined respectively for GO classification and KEGG pathway analysis.Of the total up-regulated genes,538 were annotated to 481 GO terms in the biological process category,523 were annotated to 121 GO terms in the molecular function category,and 556 were annotated to 54 GO terms in the cell group category;Of the total down-regulated genes,415 were annotated to 276 GO terms in the bioprocess category,402 were annotated to 102 GO terms in the molecular function category and 432 were annotated to 352 GO terms in the cell-group category.By KEGG pathway analysis,217 up-regulated differentially expressed genes were significantly enriched in 38 KEGG pathways and 190 down-regulated differentially.4.Alternative splicing and new transcript predictionThe alternative splicing events and new transcripts of each group were statistically analyzed.Among the 9293,15150,15925 and 12821 genes in the BF1 to BF4 sample groups,15,709,27415,29,449 and 219,900 alternative splicing events were found respectively.The number of new transcripts in each group was 1823,1889,1648 and 1794 respectively.The new transcript coding was predicted by Analyzed by CPC software,534,723,645 and 655 new transcripts were encoded in BF1 to BF4 respectively,which accounted for 29.29%,38.27%,39.14% and 36.51% respectively of the total number of new transcripts.5.Regulation of SIRT1 gene on the growth and development of bovine ovarian granulosa cells and its molecular mechanismImmunohistochemistry showed that the SIRT1 protein was expressed in granulosa cells and only in a few cell.Granulosa cells were transfected with SIRT1-bos-1597,SIRT1-bos-1471,SIRT1-bos-1186 and SIRT1-bos-1306 interfering vector,and the relative expression of SIRT1 gene in each group was detected by real-time PCR.SIRT1-bos-1471,the most potent suppressor,was transfected into granulosa cells to interfere with SIRT1 gene expression.The expression of granulosa cell proliferation,cell cycle,apoptosis and apoptosis related genes were detected at 24,36,48 and 60 h after transfection.The results showed that compared with the control group,the number of granulosa cells in the interference group was significantly increased(P<0.05);the proportion of cells in G1 phase was significantly decreased(P<0.05);the proportion of S phase cells was increased(P<0.05).The mRNA and protein expressions of Bax were significantly down-regulated(P<0.05),The mRNA and protein expressions of Bcl-2 were significantly up-regulated(P<0.05)and the secretion of estrogen and progesterone was significantly decreased(P<0.05).The results showed that SIRT1 plays an important role in the proliferation and apoptosis of granulosa cells.It is speculated that SIRT1 can inhibit the proliferation of granulosa cells by regulating the cell cycle and promote the apoptosis of granulosa cells by regulating the expression of Bcl-2 family genes.6.Interfere with INHBA gene on the growth and development of bovine ovarian granulosa cells and its regulatory mechanismImmunohistochemistry showed that INHBA protein was mainly expressed in granulosa cells and only in a very small amount of cells.The transfected granulosa cells were transfected with INHBA-bos-1365,INHBA-bos-682,INHBA-bos-603 and INHBA-bos-532.The relative expression of INHBA gene in each group was detected by real-time PCR.The best inhibitory vector INHBA-bos-682 transfected into granulosa cells TO interfere with INHBA gene expression.The expression of granulosa cell proliferation,cell cycle,apoptosis and apoptosis related genes were detected at 24,36,48 and 60 h after transfection.The results showed that compared with the control group,the number of granulosa cells in the interference group was significantly decreased(P<0.05),the proportion of cells in G1 phase was significantly increased(P<0.05),and the percentage of cells in the 60 h phase was significantly decreased(P<0.05).The apoptosis of granulosa cells was increased(P<0.05),the expression of Bax mRNA and protein were significantly increased(P<0.05),while the expression of Bcl-2 mRNA and protein(P<0.05).The secretion of estrogen and progesterone was significantly decreased(P<0.05).The results show that it plays an important role in the process of granulosa cell proliferation and apoptosis.It is speculated that INHBA promotes granulosa cell proliferation by regulating the cell cycle and inhibits granulosa cell apoptosis through the Bcl-2 pathway.In summary,the initial screening of the candidate genes that affect the development of follicles this study was conducted through the high-throughput sequencing of different diameter follicles of cattle,and the effects of SIRT1 gene and INHBA gene in follicular development and its regulatory mechanism were explored.This study lay a experimental foundation for further searching the key genes and molecular mechanisms that control follicular development. |
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