Figure 1.
Detected miRNAs in granulosa cells of DF and SF at day 7 and day of the oestrous cycle.
(A) The number of filtered sequence reads obtained in each biological replicates of SF and DF granulosa samples at day 3 and day 7 of the estrous cycle and the percentage of aligned sequences to the bovine reference genome (A). The blue and the red colour indicate the percentage of mapped and unmapped sequence reads, respectively. Rep_1, Rep_2, and Rep_3 indicate the biological replicates in each sample group. (B) Venn diagram showing the number of known miRNAs detected uniquely or commonly in SF and DF granulosa samples at day 3 and day 7 of the estrous cycle. SF Day 3 and DF Day 3 indicate the subordinate and dominant follicles, respectively at day 3, while SF Day 7 and DF Day 7 indicate the subordinate and dominant follicles, respectively at day 7 of the estrous cycle. The Venn diagram was drawn using Venny software, a freely available online tool http://bioinfogp.cnb.csic.es/tools/venny/.
Figure 2.
The top most abundant miRNAs with >2000 read counts in granulosa samples of SF or DF at day 3 and/or day 7 of the estrous cycle.
No. reads describe the average number of read counts aligned to each miRNA.
Figure 3.
Differentially expressed known miRNAs between the granulosa cells of SF and DF at day 3 of the estrous cycle.
(A) The hierarchical clustering of differentially expressed miRNAs along with their average expression difference (FC = log2 fold change), p value, and false discovery rate (FDR). Positive and negative FC values indicate up and downregulation of miRNA, respectively in SF compared to DF granulosa cells. The red and green colours designate high and low expression of miRNAs, respectively. B) The pathways enriched by genes targeted by differentially expressed miRNAs.
Figure 4.
Differentially expressed miRNAs between granulosa cells of SF and DF at day 7 of the estrous cycle.
(A) The expression patterns and hierarchical clustering of 108 differentially expressed miRNAs between granulosa cells of SF and DF. The numbers 1, 2, 3 under SF and DF indicate the biological replicates. (B) The expression patterns and hierarchical clustering of top 36 differentially expressed miRNAs along with their average expression difference (FC = log2 fold change), p values and false discovery rate (FDR). The red and green colours indicate high and low expression, respectively. Positive and negative FC values indicate up and downregulation of miRNA, respectively in SF compared to the DF granulosa cells.
Table 1.
MiRNA families co-expressed or co-repressed in granulosa cells of SF compared to DF at day 7 of estrous cycle.
Figure 5.
Significant molecular pathways (P≤0.05) enriched by genes targeted by differentially expressed miRNAs between the granulosa cells of SF and DF at day 7 of the estrous cycle.
Pathways enriched by genes potentially targeted only by miRNAs increased in SF are indicated in the left box, pathways enriched by genes potentially targeted by both miRNAs repressed and activated in SF are shown in the middle box while pathways enriched by genes potentially targeted only by miRNAs upregulated in DF but repressed in SF are described in the right box.
Figure 6.
The list of differentially expressed miRNAs whose target genes are enriched (p≤0.05) in Wnt signaling, GnRH signaling, MAPK, signaling, oocyte meiosis, TGF-beta signaling, focal, adhesion, ErbB, gap junction, axon guidance and apoptosis.
(↑) indicates increased expression while (↓) depicts the reduction of miRNA expression in granulosa cells of SF compared to DF groups at day 7 of the estrous cycle.
Figure 7.
Uniquely and commonly differentially expressed miRNAs between the granulosa cells of SF and DF at day 3 and day 7 of the estrous cycle and their pathways enriched by their potential target genes.
(↑) shows upregulation while (↓) indicate downregulation of commonly differentially expressed miRNAs in the granulosa cells of SF compared to DF at day 3 or day 7 of the estrous cycle.
Figure 8.
Scatter plot showing the read count ratio of 357 miRNAs between day 7 and day 3 in the granulosa cells of DF.
Figure 9.
The heatmaps and the hierarchical clustering depicting the expression patterns of differentially expressed miRNAs in granulosa cells of DF between day 3 and 7 of the estrous cycle.
(A) The expression patterns of miRNAs detected only at day 3 (top) or at day 7 (bottom) of the estrous cycle in granulosa cells of DF. (B) The expression patterns of miRNAs detected both at day 3 and at day 7 of the estrous cycle but significantly increased in the former group. (C) The expression patterns of miRNAs expressed both at day 3 and at day 7 of the estrous cycle but significantly increased in the later group. The colour scale shows the log2 transformed expression values. Zero colour scale indicates miRNAs with ≤1 average read count. Numbers, 1, 2 and 3 on the heatmaps describe the number of biological replicates used in each sample group. Day 3 and Day 7 indicate the stages of the estrous cycle post estrus.
Figure 10.
Graphical illustration of DF follicles and molecular pathways enriched by genes targeted by differentially expressed miRNAs between day 3 and day 7 of the estrous in granulosa cells of DF.
Pathways significantly (P≤0.05) enriched by genes potentially targeted only by miRNAs enriched at day 3 are indicated in the left box. Pathways enriched by genes potentially targeted by miRNAs increased at day 3 and day 7 of the estrous cycle are listed in the middle box and pathways enriched by genes potentially targeted only by miRNAs increased at day 7 of the estrous cycle are described in the right box. DF, dominant follicle, Day 3 and Day 7 indicate the stages of the estrous cycle post estrus.
Table 2.
MiRNA families co-expressed or co-repressed in granulosa cells of DF at day 7 compared to day 3 of the estrous cycle.
Table 3.
Novel candidate miRNAs detected in granulosa cells of SF or/and DF at day 3 or/and day 7 of the estrous cycle.
Figure 11.
The heatmap showing the qPCR data along with the deep sequencing data for randomly selected differentially expressed miRNAs.
(A) The expression pattern of candidate miRNAs in granulosa cells of SF and DF at day 3 of the estrous cycle. (B) The expression pattern of candidate miRNAs in granulosa cells of SF and DF at day 7 of the estrous cycle. (C) The expression pattern of candidate miRNAs in granulosa cells of DF at day 3 and day 7 of the estrous cycle. The red and green colours indicate high and low expression, respectively. NGS and qPCR indicate the results obtained from next generation deep sequencing and quantitative real time qPCR, respectively. Numbers, 1, 2 and 3 on the heatmaps indicate the number of biological replicates used in each sample group.
Figure 12.
The expression patterns of candidate miRNAs in theca cells of SF and DF whose granulosa cells was used for the next generation sequencing.
(A) The expression level of 9 candidate miRNAs in granulosa and theca cells of SF compared to DF at day 3 of the estrous cycle. (B) The expression level of 12 candidate miRNAs in granulosa and theca cells of SF compared to DF at day 7 of the estrous cycle. Positive and negative log2FC values indicate up and downregulation of miRNA, respectively in SF compared to the DF.