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.

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Similar to the domestic hen ovary, entry of a follicle into the preovulatory hierarchy in the turkey hen represents a process in which a single follicle initiates rapid growth and final maturation prior to ovulation. Published data derived from the laying hen support the proposal that differentiation of the follicle granulosa cell (GC) layer begins coincident with entry into the rapid growth phase and is characterized by the initial capacity for follicle stimulating hormone (FSH)-mediated cell signaling. The present studies were conducted with photostimulated B.U.T. Big 6 turkey hens to compare follicle dynamics and cellular mechanisms to those in the laying hen. The measurement and weights of turkey ovarian follicles greater than 1 mm in diameter revealed a discrete size hierarchy that was maintained throughout follicle development. GC layers collected from the single follicle initiating rapid growth (at the 11 to 13 mm stage of development) and incubated, in vitro, for 3 h with recombinant human (rh) FSH (10 ng/mL) responded with significantly increased steroidogenic acute regulatory protein (STAR) mRNA expression and progesterone production. The same treatment induced minimal STAR expression and no significant progesterone accumulation in GCs from 8 to 9 mm follicles (prior to the rapid growth phase). By comparison, dispersed GCs from 8 to 9 mm follicles pre-cultured for 18 h followed by a 3 h challenge with rhFSH resulted in significantly increased STAR expression plus progesterone production. Significantly, such cultured GCs pretreated for 15 min with transforming growth factor alpha (TGFα; 10 ng/mL) completely prevented both rhFSH-induced STAR expression and progesterone production. Culture of GCs from 8 to 9 mm follicles for 21 h with Bone Morphogenetic Protein 6 (BMP6) increased both cholesterol side-chain cleavage enzyme (CYP11A1) and FSH receptor mRNA (FSHR) expression. BMP6 also enhanced rhFSH-induced STAR expression, and this effect was blocked by TGFα. Collectively, these results support a conservation of mechanisms that maintain a hierarchy of follicles throughout development plus initiate FSH-responsiveness and GC differentiation as the recruited follicle enters the rapid growth phase in these closely related species.

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