Transcriptome Analysis on Single Small Yellow Follicles Reveals That Wnt4 Is Involved in Chicken Follicle Selection

Abstract

Ovarian follicle selection is an important process impacting the laying performance and fecundity of hens, and is regulated by follicle-stimulating hormone (FSH) through binding to its receptor [follicle-stimulating hormone receptor (FSHR)]. In laying hens, the small yellow follicle (6-8 mm in diameter) with the highest expression of FSHR will be recruited into the preovulatory hierarchy during ovarian follicle development. The study of molecular mechanism of chicken follicle selection is helpful for the identification of genes underlying egg-laying traits in chicken and other poultry species. Herein, the transcriptomes of chicken small yellow follicles differing in the mRNA expression of FSHR were compared, and a total of 17,993 genes were identified in 3 pairs of small yellow follicles. The Wnt signaling pathway was significantly enriched in the follicles with the greatest fold change in FSHR expression. In this pathway, the expression level of Wnt4 mRNA was significantly upregulated with a log₂(fold change) of 2.12. We further investigated the expression, function, and regulation of Wnt4 during chicken follicle selection and found that Wnt4 mRNA reached its peak in small yellow follicles; Wnt4 stimulated the proliferation of follicular granulosa cells (GCs), increased the expression of StAR and CYP11A1 mRNA in prehierarchical and hierarchical follicles, increased the expression of FSHR mRNA, and decreased the expression of anti-Müllerian hormone and OCLN mRNA. Treatment with FSH significantly increased Wnt4 expression in GCs. Moreover, Wnt4 facilitated the effects of FSH on the production of progesterone (P4) and the mRNA expression of steroidogenic enzyme genes in the GCs of hierarchical follicles, but inhibited the effects of FSH in the GCs of prehierarchical follicles. Collectively, these data suggest that Wnt4 plays an important role in chicken follicle selection by stimulating GC proliferation and steroidogenesis. This study provides a theoretical basis for improving the egg-laying performance of chicken and a reference for the elucidation of the molecular mechanism of follicular selection in mammals.

Keywords: Wnt4; chicken; follicle selection; follicle-stimulating hormone; granulosa cells.

Figure 1

The protein–protein interaction network of the differentially expressed genes (DEGs). STRING was used to analyze the DEGs. The network nodes represent the genes shown in Table S5 in Supplementary Material (red and blue indicate upregulated and downregulated genes, respectively) and the lines represent interactions between genes.

Figure 2

Expression of Wnt4 in follicles at different developmental stages from 30-week-old hens. (A) Expression levels of Wnt4 mRNA in 1–2 mm follicles, 6–8 mm follicles (small yellow follicles), the fifth largest follicles (F5), the third largest follicles (F3), the largest follicles (F1), and the newly postovulatory follicles (POFs). (B) Expression levels of Wnt4 protein in the various follicles. Data are presented as the mean ± SEM from at least three independent experiments. Bars with different letters are significantly different (^ABCp < 0.01).

Figure 3

Expression of Wnt4 in granulosa cells (GCs) and theca cells (TCs). (A) mRNA expression of chicken Wnt4 in the GCs and TCs of hierarchical follicles and the GCs (pre-GCs) and theca cells (pre-TCs) of prehierarchical follicles. (B) Protein expression of chicken Wnt4 in GCs, TCs, pre-GCs, and pre-TCs (^ABCp < 0.01).

Figure 4

Effect of Wnt4 overexpression on the expression of StAR and CYP11A1 in (A) pre-GCs and (B) GCs of chicken follicles (*p < 0.05, **p < 0.01, ***p ≤ 0.001).

Figure 5

Effect of Wnt4 knockdown on the expression of StAR and CYP11A1 in (A) pre-GCs and (B) GCs of chicken follicles (*p < 0.05, **p < 0.01).

Figure 6

Effect of Wnt4 on chicken granulosa cell (GC) proliferation. (A) Overexpression of Wnt4 in GCs stimulated cell proliferation, as determined by the CCK8 method. (B) Knockdown of Wnt4 in GCs inhibited cell proliferation, as determined by the CCK8 method. (*p < 0.05, **p < 0.01, ***p ≤ 0.001).

Figure 7

Effect of Wnt4 on follicle-stimulating hormone receptor (FSHR), anti-Müllerian hormone (AMH), and OCLN expression in prehierarchical follicle granulosa cells (GCs). (A) Overexpression of Wnt4 increased the expression level of FSHR, but decreased those of AMH and OCLN. (B) Knockdown of Wnt4 decreased the expression level of FSHR, but increased those of AMH and OCLN (*p < 0.05, **p < 0.01).

Figure 8

Effects of follicle-stimulating hormone (FSH) treatment on the Wnt4 mRNA and protein levels in the granulosa cells (GCs) of ovarian follicles. Various doses of FSH (0, 5, 10, and 50 ng/mL) were tested. (A) Effect of FSH on Wnt4 in the GCs of prehierarchical follicles. (B) Effect of FSH on Wnt4 in the GCs of hierarchical follicles. All data are presented as the mean ± SEM. (^abcp < 0.05; ^ABCp < 0.01).

Figure 9

Effects of Wnt4 and follicle-stimulating hormone (FSH) on mRNA expression of steroidogenic enzyme genes and steroidogenesis in prehierarchical and hierarchical follicles. (A) Effects of Wnt4 and FSH on StAR, CYP11A1, and follicle-stimulating hormone receptor (FSHR) mRNA expression and progesterone secretion in the granulosa cells (GCs) of prehierarchical follicles. Bars with different superscript letters are significantly different. (B) Effects of Wnt4 and FSH on StAR, CYP11A1, and FSHR mRNA expression and progesterone secretion in the GCs of hierarchical follicles. (^ABCp < 0.01).