Transcriptomic diversification of granulosa cells during follicular development between White Leghorn and Silky Fowl hens

Abstract

Egg production rate in chicken is related to the continuity of follicle development. In this study, we found that the numbers of white prehierarchical, dominant, and yellow preovulatory follicles in the high-yielding layer breed, White Leghorn (WL), were significantly higher than those in the low egg-yielding variety, Silky Fowl (SF). The proliferation and differentiation of granulosa cells (GCs) play an important role in follicle maturation. Histological observation revealed a large number of melanocytes in the outer granulosa layer of follicles in SF but not in WL. Finally, RNA-sequencing was used to analyze the gene expression profiles and pathways of the GC layer in the follicles in both WL and SF hens. Transcriptome analysis of prehierarchical GCs (phGCs) and preovulatory GCs (poGCs) between WL and SF showed that steroid hormone-, oxytocin synthesis-, tight junction-, and endocytosis-related genes were expressed at higher levels in WL phGCs than in SF phGCs, whereas the insulin signaling pathway- and vascular smooth muscle contraction-related genes were upregulated in SF phGCs. Fatty acid synthesis, calcium signaling, and Wnt signaling pathway-related genes were expressed at higher levels in WL poGCs than in SF poGCs; however, adrenergic signaling, cGMP-PKG, and melanogenesis-related genes were upregulated in SF poGCs. These results indicate that genes that promote GC proliferation and secretion of various sex hormones are more active in WL than in SF hens. The upregulated signaling pathways in SF help in providing energy to GCs and for angiogenesis and melanogenesis. In vitro experiments confirmed that both the proliferation of poGCs and synthesis of reproductive hormones were higher in WL than in SF hens.

Keywords: Silky Fowl; White Leghorn; granulosa cell; melanocyte; transcriptome.

FIGURE 1

Follicular morphology of WL and SF. (A) Morphology of WL prehierarchical and preovulatory follicles. (B) Morphology of SF prehierarchical and preovulatory follicles. (SWF, small white follicle; LWF, large white follicle; SYF, small yellow follicle; LYF, large yellow follicle). (C) Compare the number of WL and SF white prehierarchical follicles. (D) Compare the number of WL and SF yellow prehierarchical follicles. (E) Compare the number of WL and SF dominant follicles. (F) Compare the number of WL and SF preovulatory follicles.

FIGURE 2

Comparison of gene expression differences between WL and SF phGCs. (A) Histological observation of WL and SF phGCs. (Left is WL, right is SF. black arrow, follicle granulosa cells; red arrow, melanocytes). (B) Volcano plot of up- and down-regulated genes. (C) Top 20 KEGG pathway of up-regulated genes in SF phGCs compared with WL phGCs. (D) Top 20 KEGG pathway of down-regulated genes in SF phGCs compared with WL phGCs. The x-axis is the enrichment score, the larger the bubble, the greater the number of genes encoding differential proteins. The color of the dots represents the range of q values.

FIGURE 3

Comparison of gene expression differences between WL and SF poGCs. (A) Histological observation of WL and SF poGCs. (Left is WL, right is SF. black arrow, follicle granulosa cells; red arrow, melanocytes). (B) Volcano plot of up- and down-regulated genes. (C) Top 20 KEGG pathway of up-regulated genes in SF poGCs compared with WL poGCs. (D) Top 20 KEGG pathway of down-regulated genes in SF poGCs compared with WL poGCs. The x-axis is the enrichment score, the larger the bubble, the greater the number of genes encoding differential proteins. The color of the dots represents the range of q values.

FIGURE 4

Quantitative real-time PCR validation of differentially expressed genes identifed in transcriptome sequencing.

FIGURE 5

Comparison of proliferation and differentiation between WL and SF poGCs cultured in vitro. (A) Morphology of WL poGCs cultured in vitro (1–6 days). (B) Morphology of SF poGCs cultured in vitro (1–6 days). (C) Comparison of WL poGCs and SF poGCs cell proliferation ability. (D) Expression of genes associated with the synthesis of sex hormones and receptors in GCs were detected. (E) Comparison of secretion FSH, LH and P4 between WL poGCs and SF poGCs. (*p < 0.05; **p < 0.01; ***p < 0.001).