Differentiation of the granulosa layer from hen prehierarchal follicles associated with follicle‐stimulating hormone receptor signaling

Abstract

Recruitment of a single follicle into the preovulatory hierarchy of the domestic hen ovary occurs from a small cohort of prehierarchal follicles measuring 6-8 mm in diameter. We have previously reported that granulosa cells (GCs) collected from prehierarchal follicles express highest levels of membrane-localized follicle-stimulating hormone receptor (FSHR) during follicle development, yet fail to initiate signaling via cAMP following short-term incubation with FSH. Consequently, GC from prehierarchal follicles remain in an undifferentiated state and lack the capacity for steroidogenesis due to a deficiency of cAMP-dependent STAR protein and CYP11A1 gene expression. The present studies investigate FSH responsiveness in GC before and after the transition from undifferentiated to a differentiated state at follicle recruitment. Before recruitment focus is directed toward the inhibition of FSHR-signaling by β-ARRESTIN (βARR). Specifically, knockdown of βARR messenger RNA in cultured, undifferentiated GC using small interfering RNA facilitated FSH-induced cAMP formation, STAR expression, and progesterone production. Furthermore, overexpression of bovine βARR1 and G-protein-coupled receptor kinase2 in actively differentiating GC significantly decreased cAMP accumulation and progesterone production following a challenge with FSH. We propose that a βARR-mediated mechanism maintains FSHR unresponsiveness in undifferentiated GC from prehierarchal follicles, and as a result prevents GC differentiation until the time of follicle recruitment.

Regulation of vasoactive intestinal peptide receptor (VPAC) signaling in Undifferentiated hen granulosa cells

“…Among the reported actions of VIP within the GC layer from preovulatory follicles are included the stimulation of cyclic adenosine monophosphate (cAMP) production, which, among other cellular functions, promotes steroidogenic acute regulatory protein (STAR) expression and progesterone production Johnson et al, , plus increases messenger RNA (mRNA) encoding the biological clock protein BMAL. Nevertheless, as recently reported, regarding another G‐protein‐coupled receptor, follicle‐stimulating hormone (FSH; Kim & Johnson, ), VIP‐induced cell signaling via cAMP within the hen GC layer is not initiated until the time of follicle recruitment into the preovulatory hierarchy (Kim & Johnson, ). Notably, undifferentiated hen GCs collected from prehierarchal (6–8 mm) follicles and incubated with FSH in media alone for 3 hr acquire FSH‐responsiveness, whereas hen GCs incubated in media containing FSH in the presence of a mitogen‐activated protein kinase (MAPK) ligand (e.g., transforming growth factor α) remain in an undifferentiated state (Kim & Johnson, ; Li & Johnson, ).…”

“…Nevertheless, as recently reported, regarding another G‐protein‐coupled receptor, follicle‐stimulating hormone (FSH; Kim & Johnson, ), VIP‐induced cell signaling via cAMP within the hen GC layer is not initiated until the time of follicle recruitment into the preovulatory hierarchy (Kim & Johnson, ). Notably, undifferentiated hen GCs collected from prehierarchal (6–8 mm) follicles and incubated with FSH in media alone for 3 hr acquire FSH‐responsiveness, whereas hen GCs incubated in media containing FSH in the presence of a mitogen‐activated protein kinase (MAPK) ligand (e.g., transforming growth factor α) remain in an undifferentiated state (Kim & Johnson, ; Li & Johnson, ). More recently, we provided evidence that before follicle recruitment, GC signaling via the FSH receptor is precluded by a β‐ARRESTIN ( βARR )‐mediated mechanism (Kim & Johnson, ) and have proposed that this differentiation‐inhibiting process is maintained, at least in part, by MAPK signaling.…”

“…Notably, undifferentiated hen GCs collected from prehierarchal (6–8 mm) follicles and incubated with FSH in media alone for 3 hr acquire FSH‐responsiveness, whereas hen GCs incubated in media containing FSH in the presence of a mitogen‐activated protein kinase (MAPK) ligand (e.g., transforming growth factor α) remain in an undifferentiated state (Kim & Johnson, ; Li & Johnson, ). More recently, we provided evidence that before follicle recruitment, GC signaling via the FSH receptor is precluded by a β‐ARRESTIN ( βARR )‐mediated mechanism (Kim & Johnson, ) and have proposed that this differentiation‐inhibiting process is maintained, at least in part, by MAPK signaling. The current study examined the ability of a small interfering RNA (siRNA) directed against Gallus βARR to prevent VIP‐induced VPAC signaling via cAMP in GC from 6 to 8 mm prehierarchal follicles, and consequently, initiate or further increase expression of several notable markers of GC differentiation.…”

“…Undifferentiated GCs collected from 6 to 8 mm prehierarchal follicles were transfected with a scrambled sequence (Scr) or a siRNA directed against Gallus βARR (Table ) as previously reported (Kim & Johnson, ). After overnight culture, replicate sets of Scr‐ or siRNA‐transfected GCs were pretreated with 3‐isobutyl‐1‐methylxanthine (10 μM), then one set of cells transfected with Scr or siRNA was challenged for 4 hr without or with chicken VIP (1 μM) (Kim & Johnson, ) whereas the second set served as the control.…”

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

“…Apparently, this process seems to be complex and highly coordinated, involving a number of divergent biological effects on the maturation of oocytes and differentiation and proliferation of granulosa and theca cells within the ovarian follicles [8]. In addition, the follicle-stimulating hormone (FSH) has been implicated in facilitating the recruitment of follicles [6,7,[9][10][11][12]. Similarly, follicle stimulating hormone receptors (FSHR) are reportedly expressed on the granulosa layer of developing follicles, and mRNA expression of these receptors (FSHR) changes with progressing follicular maturation [3,6,13].…”