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.

Effect on physiological and production parameters upon supplementation of fermented yeast culture to Nicobari chickens during and post summer

“…In pigeons, it has been reported that, heat stress elevates plasma leptin (Al‐Azraqi, 2008). Furthermore, its expression at the level of the ovary (Niu et al, 2019 ) suggests that Leptin might directly act on the ovary to regulate chicken reproductive function. A higher concentration of plasma Leptin in the control group might have caused a decrease in the percentage of egg production (Lei et al, 2014).…”

“…Leptin hormone gene has been localized in chickens in other tissues including brain (Brown et al, 2009; Serrousi et al, 2017). It is known that leptin and GH regulate ovarian follicle development (Niu et al, 2019; Socha & Hrabia, 2019). It is reported that enhancement of leptin receptor signalling leads to reduction in follicle development, ovarian hormone secretion and further production performance (Lei et al, 2014).…”

Leptin Modulates the mRNA Expression of Follicle Development Markers in Post-hatch Chicks in an Age-Dependent Manner

“…For a leptin administration study, recombinant mouse leptin (rmleptin; Sigma–Aldrich Japan, Tokyo, Japan) was used in this study, because genuine recombinant chicken leptin derived from recently cloned cDNA (Farkašová et al, 2016 ; Seroussi et al, 2016 ) may not be available. On the other hand, rmleptin remains biologically active in chicken and other birds and is used in ovarian function and other studies (Bungo et al, 1999 ; Máčajová et al, 2002 , 2003 ; Lamošová et al, 2003 ; Song et al, 2009 ; Adachi et al, 2012 ; Hu et al, 2012 ; Niu et al, 2019 ). The rmleptin was dissolved in phosphate-buffered saline [PBS (–)] and injected intraperitoneally (i.p.)…”

Morphometric Evaluation of Spermatogenic Cells and Seminiferous Tubules and Exploration of Luteinizing Hormone Beta Polypeptide in Testis of Datong Yak

“…qPCR was used to determine the expression pattern of LHB. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) and beta actin (ACTB) were used as reference genes [20,21]. qPCR was performed with 12.5 µL TB Green TM Premix Ex Taq TM II (2×) (TaKaRa Bio Inc., Shiga, Japan).…”