Initiation of follicular atresia: gene networks during early atresia in pig ovaries

in Reproduction
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In mammals, more than 99% of ovarian follicles undergo a degenerative process known as atresia. The molecular events involved in atresia initiation remain incompletely understood. The objective of this study was to analyze differential gene expression profiles of medium antral ovarian follicles during early atresia in pig. The transcriptome evaluation was performed on cDNA microarrays using healthy and early atretic follicle samples and was validated by quantitative PCR. Annotation analysis applying current database (Sus scrofa 11.1) revealed 450 significantly differential expressed genes between healthy and early atretic follicles. Among them, 142 were significantly upregulated in early atretic with respect to healthy group and 308 were downregulated. Similar expression trends were observed between microarray data and quantitative RT-PCR confirmation, which indicated the reliability of the microarray analysis. Further analysis of the differential expressed genes revealed the most significantly affected biological functions during early atresia including blood vessel development, regulation of DNA-templated transcription in response to stress and negative regulation of cell adhesion. The pathway and interaction analysis suggested that atresia initiation associates with (1) a crosstalk of cell apoptosis, autophagy and ferroptosis rather than change of typical apoptosis markers, (2) dramatic shift of steroidogenic enzymes, (3) deficient glutathione metabolism and (4) vascular degeneration. The novel gene candidates and pathways identified in the current study will lead to a comprehensive view of the molecular regulation of ovarian follicular atresia and a new understanding of atresia initiation.


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    To validate the microarray result, gene expression levels of three downregulated genes including VEGFA (A), CYP19A1 (B), STAR (C) and three upregulated genes including RSAD2 (D), ALCAM (E) and CYP11A1 (F) were detected by qRT-PCR in H, EA and PA groups. Note: The different small letters indicate P < 0.05. EA, early atresia; H, healthy; PA, progressive atresia; qRT-PCR, quantitative RT-PCR.

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    Gene expression levels of four apoptosis-related genes including FASLG (A), TP53 (B), CASP3 (C) and BAK1 (D) were detected by qRT-PCR. Significantly increases were observed in PA group with respect to H and EA groups. Note: The different small letters indicate P < 0.05. EA, early atresia; H, healthy; PA, progressive atresia; qRT-PCR, quantitative RT-PCR.

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    GO analysis was performed using DEGs between H and EA follicles. The 2D bar graph of biological process (A), cellular components (B) and molecular functions (C) categories was generated by ClueGO. The vertical axis shows terms GO categories and the horizontal axis shows the percentage of DEGs out of all genes included in each term. Numbers in the end of each bar represent number of DEGs. Terms with same color represents groups consisting of terms with interrelations. DEG, differentially expressed genes.

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    Bubble chart of potential signaling pathways generated by DEGs. Pathway analysis was performed to associate the unique DEGs with pathways using the KEGG database. The size and color of each bubble represent number of DEGs in each pathway and P value respectively. DEG, differentially expressed genes.

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    Main pathways and interactions. Pathway analysis was performed using DEGs. Each solid circle represents an individual pathway and the size of each circle indicates number of DEGs included in the pathway. Circles that connected by straight lines represent pathway interactions. DEG, differentially expressed genes.


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