Proteome profiling of chicken ovarian follicles immediately before and after cyclic recruitment.

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.

Response of hen pre-recruitment ovarian follicles to follicle stimulating hormone, in vivo.

In laying hens, pre-recruitment ovarian follicles (1-8 mm diameter) are arranged as a continuum of size and predicted maturity. Cyclic recruitment of a pre-recruitment follicle to the preovulatory stage begins, in part, by the ability of the granulosa cell (GC) layer to initiate responsiveness to follicle stimulating hormone- (FSH-) induced cyclic adenosine monophosphate. The objective of this study was to determine if increased circulating concentrations of FSH during the ovulatory cycle increase the number of recruited follicles, in a dose-dependent manner. Equine chorionic gonadotropin (eCG) was initially tested due to its FSH-like properties and long half-life. Laying hens were injected, i.m., with 0 or 100 IU eCG, and ovaries were collected 29 h later. Recruited follicles were initially identified based on incorporation of yellow yolk and a weight of 250-900 mg. Recruitment was subsequently confirmed by both incubating the GC layer for 3 h with recombinant human (rh) FSH to establish FSH-responsiveness and quantifying P450 side-chain cleavage enzyme (CYP11A1) mRNA. Additional hens were injected with 0, 30, 75, and 300 IU eCG to establish a dose-response. Because eCG exhibits some luteinizing hormone activity, FSH-induced recruitment was evaluated by injecting 0.1, 0.33, 0.66, 1 or 3.3 µg rhFSH. Ovaries were collected 29 h post-injection, and expression of CYP11A1 mRNA was quantitated in GCs from recruited and pre-recruitment follicles. One hundred IU eCG induced recruitment of 2-8 follicles compared to a single follicle in control hens. In contrast to pre-recruitment follicles, incubated GC from eCG-recruited follicles had initiated differentiation, indicated by increased CYP11A1 and rhFSH-induced STAR mRNA and progesterone. Equine CG and rhFSH each increased the number of recruited follicles in a dose-dependent manner. Further, CYP11A1 mRNA was significantly increased in GC layers from recruited, compared to non-recruited, follicles. We conclude that FSH-responsiveness within the GC layer of each pre-recruitment follicle increases with follicle size, and propose that this establishes the order of daily follicle recruitment.

Relationship between cyclic follicle recruitment and ovulation in the hen ovary.

Determining whether follicle recruitment in the domestic hen is functionally linked to ovulation would inform investigations on the exact time of cyclic recruitment and subsequently, the earliest cellular event mediating recruitment. The objective of the present studies was to determine if the absence of ovulation results in the failure of cyclic recruitment. 3 groups of Hy-Line W-36 hens were studied: no-ovulation, first-ovulation, or late-ovulation within the sequence. Time and occurrence of oviposition and ovulation were documented by video recording and cloacal palpation. To determine the presence of a recently recruited follicle, ovaries were collected during the last hour of photoperiod, and follicles weighing 280 to 900 mg, with yellow yolk incorporated, and diameter of 9 to 12 mm, were considered recently recruited. To compare the amount of yolk uptake in recruited follicles, hens were fed Sudan IV dye 6 h before ovary collection. Percent dyed yolk was quantified from cross-sections of the recruited follicles. To confirm follicle viability, granulosa cell (GC) mitotic activity was assessed via flow cytometry. Initial results indicated the presence of a recently recruited follicle in 10 of 11, 7 of 10, and 9 of 10, no-ovulation, first-ovulation, and late-ovulation ovaries, respectively, with no significant differences in weight (543 ± 62 mg, 456 ± 71 mg, 620 ± 75 mg, respectively). There were no significant differences in yolk incorporation among the most recently recruited follicles with 19.0 ± 2.0%, 18.0 ± 3.7%, and 19.8 ± 3.2% dyed yolk in no-ovulation, first-ovulation, and late-ovulation ovaries, respectively. Finally, there were no significant differences in % GC in the S/G2-M phase of mitosis among recruited follicles (19.0 ± 5.0%, 22.0 ± 4.2%, 15.67 ± 1.0%, in no-ovulation, first-ovulation, and late-ovulation ovaries, respectively), confirming viability of all recruited follicles. We conclude that cyclic recruitment occurs independently of ovulation and propose that the order and timing of cyclic recruitment is predetermined at an earlier stage of follicle development.

Lack of a relationship between plasma fibroblast growth factor-23 and phosphate utilization in young chicks.

Fibroblast growth factor 23 (FGF-23) is proposed to be the hormone that controls phosphate (P) homeostasis in chickens. This study was initiated to investigate the effect of feeding young chicks diets that were either adequate (0.45%) or marginal (0.25%) in available P content on plasma FGF-23 levels. The dietary level of available P significantly (P ≤ 0.05) affected bone mineralization and bone length, but was without effect (P > 0.05) on growth rate and circulating FGF-23 concentrations. Substantial individual variation in bone mineralization and plasma FGF-23 levels was observed, and the correlation between these two variables was non-significant (P > 0.05). This suggested that there was no alteration in FGF-23 activity in response to suboptimal dietary P intake. The relationship of these observations to studies on the immunosuppression of FGF-23 activity is subsequentlydiscussed.

Follicle dynamics and granulosa cell differentiation in the turkey hen ovary.

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.