Systematic Analysis of Long Noncoding RNA and mRNA in Granulosa Cells during the Hen Ovulatory Cycle

Abstract

Long non-coding RNAs (lncRNAs) and mRNAs are temporally expressed during chicken follicle development. However, follicle transcriptome studies in chickens with timepoints relating to changes in luteinizing hormone (LH) levels are rare. In this study, gene expression in Rohman layers was investigated at three distinct stages of the ovulatory cycle: zeitgeber time 0 (ZT0, 9:00 a.m.), zeitgeber time 12 (ZT12, 9:00 p.m.), and zeitgeber time 20 (ZT20, 5:00 a.m.) representing the early, middle, and LH surge stages, respectively, of the ovulatory cycle. Gene expression profiles were explored during follicle development at ZT0, ZT12, and ZT20 using Ribo-Zero RNA sequencing. The three stages were separated into two major stages, including the pre-LH surge and the LH surge stages. A total of 12,479 mRNAs and 7528 lncRNAs were identified among the three stages, and 4531, 523 differentially expressed genes (DEGs) and 2367, 211 differentially expressed lncRNAs (DELs) were identified in the ZT20 vs. ZT12, and ZT12 vs. ZT0, comparisons. Functional enrichment analysis revealed that genes involved in cell proliferation and metabolism processes (lipid-related) were mainly enriched in the ZT0 and ZT12 stages, respectively, and genes related to oxidative stress, steroids regulation, and inflammatory process were enriched in the ZT20 stage. These findings provide the basis for further investigation of the specific genetic and molecular functions of follicle development in chickens.

Keywords: chicken; granulosa cell; lncRNA; luteinizing hormone surge; ovulation.

Figure 1

Changes in serum luteinizing hormone (LH) level and H&E-stained follicle granulosa layer among three stages of the ovulatory cycle. (a) LH concentration was measured in chicken serum during the ovulatory cycle (4-h intervals from ZT0 to ZT24 and six repetitions for each stage), and the highest LH level was detected at the ZT20 stage. (b) Chicken F1 follicles were embedded in paraffin and stained with hematoxylin and eosin (H&E) at ZT0, ZT12, and ZT20. ZT0 (9:00 a.m.), ZT12 (9:00 p.m.), and ZT20 (5:00 a.m.) represent the early, middle, and LH surge stages, respectively, of the ovulatory cycle.

Figure 2

Global gene expression profiles of the F1 follicle during development. Hierarchical clustering of all samples (a) and t-SNE plot (b) of lncRNAs and mRNAs based on expression profiles in different development stages. The top and left panel is the sample and gene tree, and the value represents the log2 transformed values of (TPM + 1). ZT0 (9:00 a.m.), ZT12 (9:00 p.m.), and ZT20 (5:00 a.m.) represent the early, middle, and LH surge stages, respectively, of the ovulatory cycle.

Figure 3

Gene ontology (GO) and pathway analysis of differentially expressed genes (DEGs) during follicle development in the chicken ovulatory cycle. (a) Number of DEGs in pairwise stages. (b) Enriched GO terms (top) and (c) pathways (bottom) of DEGs in ZT12 vs. ZT0 group. (d) Enriched GO terms (left) and (e) pathways (right) of DEGs in ZT12 vs. ZT0 group. Directly up-regulated (red) and down-regulated (blue) gene numbers and ontologies are shown.

Figure 4

Gene ontology (GO) and pathway analysis of differentially expressed genes (DEGs) during follicle development in the chicken ovulatory cycle. (a) Number of DELs in pairwise stages. (b) Enriched GO terms (up) and pathways (bottom) of DEGs in ZT20 vs. ZT12 group. The up-regulated (red) and down-regulated (blue) gene numbers and gene ontology terms are shown.

Figure 5

Expression patterns of the time-series clusters. (a) STEM analysis identified the temporal expression profiles of differentially expressed genes (DEGs) and differentially expressed lncRNAs (DELs) with p < 0.05. The profile number in the top left corner of each profile box was assigned by STEM. The number in the bottom left represents the adjusted p-value. (b) A heatmap of the transcripts’ expression in cluster 10 based on normalized data of expression values. (c) Enriched GO terms (circle) and pathways (triangle) of DEGs in the cluster 10 module.

Figure 6

Expression and function of lncRNA G13560 in follicle development during the chicken ovulatory cycle. (a) Expression of genes associated with steroid regulation was up-regulated in the ZT20 stage compared with the other two stages. (b) lncRNA was co-expressed with these steroid-related genes and displayed a similar expression pattern. (c) lncRNA G13560 expression was confirmed by qRT-PCR among the three development stages. Data are shown as mean ± SD. (d) Co-expression of lncRNA and mRNA was validated in cultured granulosa cells with or without LH treatment. STAR, steroidogenic acute regulatory protein; GAPDH was used as a control. Data are means ± SD and are representative of at least three independent experiments. * p < 0.05. ZT0 (9:00 a.m.), ZT12 (9:00 p.m.), and ZT20 (5:00 a.m.) represent the early, middle, and LH surge stages, respectively, of the chicken ovulatory cycle.

Figure 7

qPCR verification of differentially expressed genes (DEGs) and differentially expressed lncRNAs (DELs) during follicle development. (a) Expression patterns of three DELs (G17003, G5405, and G4501) and three DEGs (LRP8, TRAF2, and TTYH3) were validated by qPCR. The x-axis represents the three developmental stages during the ovulatory cycle. The y-axis represents the relative expression levels of each gene; the left blue color represents relative expression by qPCR, and the right red color is the value of RNA-seq data with log2(TPM + 1). Data are shown as means ± SD and are representative of at least three independent experiments. (b) Transcriptome-wide RNA sequencing dynamics to reveal main functional factors of granulosa cell during follicle development. ZT0 (9:00 a.m.), ZT12 (9:00 p.m.), and ZT20 (5:00 a.m.) represent the early, middle, and LH surge stages, respectively, of the chicken ovulatory cycle.