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Simple Summary  In highly efficient laying hens, such as in commercial layer lines, the development of follicles is mainly characterized by an organized follicular hierarchy. Leptin has been implicated in the modulation of female reproduction in vertebrate animals.  Forkhead box L2  ( FOXL2 ) and  R-spondin1 (RSPO1)  have also been implicated in the regulation of ovarian functions and the development of follicles. In this study, using a laying hen model, we observed abundant mRNA expression of  FOXL2 and RSPO1  in small (prehierarchical) and large (hierarchical) follicles, respectively.  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. However, theca cells exhibited a significantly higher mRNA expression of  RSPO1  in F4 to F1 follicles. In subsequent experiment, we observed that 100 ng/mL leptin significantly modulated  FOXL2  and  RSPO1  expression in cultured granulosa cells harvested from large hierarchical and small prehierarchical follicles. These findings reasonably suggest that  FOXL2  and  RSPO1  genes may have a role in modulating the ovarian mechanisms, possibly affecting follicle growth and selection in laying hens. In addition, we demonstrated that leptin administration to granulosa cells in vitro modulated  FOXL2  and  RSPO1  expression, suggesting an implication of leptin in the follicular development and steroidogenesis in laying hens. However, further focused studies are warranted to improve our understanding of the exact roles played by these genes in follicle development and selection in laying hens.    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.

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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