Anthony L. Johnson

A shotgun proteomics study using isobaric tags for relative and absolute quantification labeling was conducted to characterize proteins in chicken ovarian follicles immediately before and after cyclic recruitment. Granulosa cell (GC) layers from the most recently recruited follicle (GC9) and from each of the four largest prerecruitment follicles (GC1–4) plus theca tissue (TH) from the most recently recruited (TH9) and largest prerecruitment (TH1) follicles were compared. Of 1535 proteins identified, none were determined to be differentially expressed between TH9 and TH1. A pairwise comparison between GC9 and GC1, GC2, GC3, or GC4 resulted in one, five, five, and six differentially expressed proteins, respectively, including yolk and cholesterol transport proteins (vitellogenin 1–3 and apolipoprotein B). In addition, transforming growth factor‐beta 1 and microRNA‐21 pathways were predicted to be activated at recruitment. We also report, for the first time, the expression of the neuropeptide, RELAXIN‐3 (RLN3), in GC. Quantitative polymerase chain reaction determined RLN3 expression to be highest in GC9 and GC1, but its receptors, RXFP1 and RXFP3, were highest in TH and ovarian stroma, respectively. Overall, cyclic recruitment is associated with changes in protein expression predominantly within follicle GC, and a potential role for RLN3 in follicle recruitment and the initiation of GC differentiation warrants further investigation.

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.

SUMMARY: 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.

In the hen ovary, selection of a follicle into the preovulatory hierarchy occurs from a small cohort of prehierarchal (6–8 mm) follicles. Prior to follicle selection the granulosa layer remains in an undifferentiated state despite elevated follicle-stimulating hormone receptor (FSHR) expression. The present studies describe a role for bone morphogenetic protein 4 (BMP4) in supporting FSHR mRNA expression in granulosa cells from prehierarchal follicles and promoting differentiation at follicle selection. Culture of undifferentiated granulosa cells in culture medium alone resulted in a significant decline in levels of FSHR mRNA (by ∼80% compared to freshly collected cells). By comparison, granulosa cultured with BMP4 (10–100 ng/ml) maintained FSHR and expression at approximately in vivo levels. Because both granulosa and theca tissues from prehierarchal follicles express BMP4, it is suggested that BMP4 acts in a paracrine and/or autocrine fashion to support elevated FSHR expression prior to follicle selection. Granulosa cells cultured with BMP4 for 24 h also initiated FSH-induced cAMP production and indirectly initiated anti-Mullerian hormone (AMH), CYP11A, and STAR expression plus progesterone production. However, pretreatment with the BMP antagonist NOGGIN or the mitogen-activated protein kinase (MAPK) agonist transforming growth factor alpha attenuated or blocked each action promoted by BMP4. We conclude that prior to and immediately after selection, BMP4 serves to support FSHR expression within the granulosa layer, yet prior to selection, multiple factors (including inhibitory MAPK signaling, AMH, and BMP antagonists) can modulate FSHR expression and suppress FSH-mediated cell signaling to prevent granulosa cell differentiation prior to follicle selection.