Vasoactive intestinal peptide promotes differentiation and clock gene expression in granulosa cells from prehierarchal follicles

Abstract

Vasoactive intestinal peptide (VIP) signaling via cyclic adenosine monophosphate (cAMP) is reported to stimulate steroidogenesis in ovarian granulosa cells from a variety of vertebrate species, including the domestic hen. Prior to follicle selection in the hen ovary (i.e., cyclic recruitment) follicle-stimulating-hormone (FSH)-induced cAMP signaling is absent within the granulosa layer until immediately following follicle selection. As a consequence, granulosa cells remain in an undifferentiated state and are unable to initiate FSH-induced steroidogenesis. VIP receptors (VPAC1 and VPAC2), like the FSH receptor, are G protein-coupled receptors, so we predicted that VIP signaling in granulosa cells is also absent in follicles that have not yet been selected into the preovulatory hierarchy. Initial studies established that mRNA encoding VPAC1 and VPAC2 are expressed within the granulosa cells throughout follicle development. Nevertheless, undifferentiated granulosa cells from prehierarchal (6-8 mm) follicles do not accumulate cAMP in response to a 4-hr incubation with chicken VIP; the capacity for such receptor signaling is attained only following selection within actively differentiating granulosa cells. VIP treatment did, however, increase expression of mRNA encoding the Gallus circadian clock protein, BMAL1-but only in granulosa cells collected from selected follicles. These findings provide evidence that, at follicle selection, the acquisition of VIP-induced cAMP cell signaling helps initiate and promote the differentiation of of granulosa cells. Furthermore, we propose that VIP signaling may regulate BMAL1 expression and, thus, a daily rhythmicity within granulosa cells of preovulatory follicles. Mol. Reprod. Dev. 83: 455-463, 2016. © 2016 Wiley Periodicals, Inc.

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

“…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 1) as previously reported (Figure 2a); this particular circadian clock-related protein is associated with initiating and phasing the daily rhythmicity of cellular functions within GC from quail preovulatory follicles (Nakao, Yasuo, Nishimura et al, 2007). By comparison, neither PER2 nor CLOCK mRNA was previously found to be increased after incubation for 4 hr with VIP (Kim & Johnson, 2016), and neither PER2 nor CLOCK was upregulated by VIP following the knockdown of βARR (Figure 2b,c).…”

“…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. Not unexpectedly, and because GCs were cultured in the absence of any MAPK ligand, VIP treatment increased levels of cAMP, progesterone production, and expression of STAR protein in both Scr‐ and siRNA‐transfected cells compared with Scr or siR cells without VIP treatment ( p < 0.05).…”

“…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, 1994, 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; , 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, 2016). 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 Li & Johnson, 1993).…”

“…T A B L E 1 siRNA Sequences for Gallus β-ARRESTIN1 and the nontargeting control Target Sequence β-ARRESTIN1 Sense 5′-CCAACAAGACGGUGAAGAAGAUCAA-3′ Antisense 5′-UUGAUCUUCUUCACCGUCUUGUUGGUG-3′ Scrambled (Scr) Sense 5′-CGUUAAUCGCGUAUAAUACGCGUAT-3′Antisense 5′-AUACGCGUAUUAUACGCGAUUAACGAC-3′Note. siRNA: small interfering RNA.Scr-or siRNA-transfected GCs were pretreated with 3-isobutyl-1methylxanthine (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, 2016) whereas the second set served as the control. Not unexpectedly, and because GCs were cultured in the absence of any MAPK ligand, VIP treatment increased levels of cAMP, progesterone production, and expression of STAR protein in both Scr-and siRNA-transfected cells compared with Scr or siR cells without VIP treatment (p < 0.05).…”

Grand and Less Grand Challenges in Avian Physiology

“…When the mature follicle (F 1 ) is ovulated, an additional follicle(s) are recruited to the hierarchy. Alan Johnson's laboratory has conducted a series of elegant studies defining the roles of hormones, neuropeptides and growth factors [luteinizing hormone (LH), follicle stimulating hormone (FSH), vasoactive intestinal peptide (VIP), epidermal growth factor receptor ligands (EGFRL), transforming growth factor β (TGF β) and bone morphogenetic proteins (BMP4/6)] on different sized follicles in the chicken ovary (e.g., Ocón-Grove et al, 2012; Kim and Johnson, 2016; reviewed Johnson, 2015). Recently, the role of epigenetic changes within the follicles has begun to be elucidated (Zhu et al, 2015).…”