Expression of FOXL2 and RSPO1 in Hen Ovarian Follicles and Implication of Exogenous Leptin in Modulating Their mRNA Expression in In Vitro Cultured Granulosa Cells

Abstract

In this study, using a laying hen model, we determined the expression of FOXL2 and RSPO1 in different central and peripheral tissue and ovarian follicles at different stages of development. At the same time, mRNA expression of both genes in granulosa and theca cells harvested from follicles at different stages of folliculogenesis was also evaluated. Finally, we assessed the effect of leptin treatment on expression of FOXL2 and RSPO1 in in vitro cultured granulosa cells harvested from 1-5 mm to F3-F1 follicles. Our RT-qPCR results revealed that a comparatively higher expression of FOXL2 and RSPO1 was observed in ovary, hypothalamus, and pituitary. Abundant mRNA expression of FOXL2 was observed in small prehierarchical follicles (1-1.9 and 2-2.9 mm follicles; p < 0.05), whereas mRNA expression of RSPO1 showed an increasing trend in large hierarchical follicles (F5-F1), and its abundant expression was observed in post-ovulatory follicles. FOXL2 mRNA expression was stable in granulosa cells harvested from 3-5 mm to F4 follicles, and exhibited a significantly higher expression in large hierarchical follicles. Conversely, relatively low mRNA expression of FOXL2 was observed in theca cells. RSPO1 mRNA expression was relatively lower in granulosa cells; however, theca cells exhibited a significantly higher mRNA expression of RSPO1 in F4 to F1 follicles. In the next experiment, we treated the in vitro cultured granulosa cells with different concentrations (1, 10, 100, and 1000 ng/mL) of exogenous leptin. Compared to the control group, a significant increase in the expression of FOXL2 was observed in groups treated with 1, 10, and 100 ng/mL leptin, whereas expression of RSPO1 was increased in all leptin-treated groups. When treated with 100 ng/mL leptin, FOXL2 and RSPO1 expression was upregulated in cultured granulosa cells harvested from both large hierarchical (F3-F1) and small prehierarchical follicles (1-5 mm). Based on these findings and evidence from mainstream literature, we envisage that FOXL2 and RSPO1 genes (in connection with hypothalamic-hypophysis axis) and leptin (via modulation of FOXL2 and RSPO1 expression) might have significant physiological roles, at least in part, in modulating the ovarian mechanisms, such as follicle development, selection, and steroidogenesis in laying hens.

Keywords: FOXL2; RSPO1; granulosa cells; hierarchical follicles; laying hen; leptin; ovary; prehierarchical follicles; theca cells.

Figure 1

The mRNA expression of FOXL2 and RSPO1 in different tissues of laying hens. (A) FOXL2 mRNA expression relative to GAPDH and ACTB mRNA. (B) RSPO1 mRNA expression relative to GAPDH and ACTB. Values represent mean ± SEM, and bars with different superscripts are significantly different (p < 0.05). Data were analyzed using one-way ANOVA followed by post-hoc Duncan’s test.

Figure 2

Expression of FOXL2 (A) and RSPO1 (B) mRNA in hen ovarian follicles at different stages of development. The mRNA expression levels are relative to GAPDH and ACTB. Bars (mean ± SEM) with different letters are significantly different (p < 0.05). Data were analyzed using one-way ANOVA followed by post-hoc Duncan’s test. Numbers in the x-axis (i.e., 1–1.9, 2–2.9, 3–3.9, 4–4.9, 5–5.9, 6–6.9, 7–7.9, and 8–9) show prehierarchical follicles grouped according to diameter (mm): 1–9 mm, prehierarchical follicles; F5 to F1, hierarchical follicles; POF, post-ovulatory follicles.

Figure 3

The mRNA expression of FOXL2 (A) and RSPO1 (B) in theca and granulosa cells harvested from follicles at different stages of development. Bars with different lowercase letters are significantly different between the same types of cells from follicles of different sizes (p < 0.05). Note: * and ** indicate significant differences between theca and granulosa cells in follicles of the same size (p < 0.05 and p < 0.01, respectively). Data were analyzed using one-way ANOVA followed by post-hoc Duncan’s test. The mRNA expression levels are relative to GAPDH and ACTB.

Figure 4

Effect of leptin (at different concentrations) on mRNA expression of FOXL2 (A) and RSPO1 (B) in cultured chicken granulosa cells. Values (mean ± SEM) with different lowercase letters are significantly different (p < 0.05). Data were analyzed using one-way ANOVA followed by post-hoc Duncan’s test. Values in the x-axis (0, 1, 10, 100, and 1000) represent different concentrations (ng/mL) of leptin. The mRNA expression levels are relative to GAPDH and ACTB.

Figure 5

Effect of leptin (100 ng/mL) on mRNA expression of FOXL2 (A) and RSPO1 (B) in granulosa cells harvested from large hierarchical (F3–F1) and small prehierarchical follicles (1–5 mm). Values (mean ± SEM) with different lowercase letters within the same group are significantly different (p < 0.05). Data were analyzed using a Student’s t-test. The mRNA expression levels are relative to GAPDH and ACTB. Definitions: treated indicates granulosa cells treated with 100 ng/mL leptin; blank indicates control group without leptin addition.