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.

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

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 ß-ARRESTIN (ßARR). Specifically, knockdown of ßARR messenger RNA in cultured, undifferentiated GC using small interfering RNA facilitated FSH-induced cAMP formation, STAR expression, and progesterone production. Furthermore, overexpression of bovine ßARR1 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 ßARR-mediated mechanism maintains FSHR unresponsiveness in undifferentiated GC from prehierarchal follicles, and as a result prevents GC differentiation until the time of follicle recruitment.

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

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.

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.

Anti-Müllerian hormone (AMH) receptor type II expression and AMH activity in bovine granulosa cells.

Anti-Müllerian hormone (AMH) produced by granulosa cells has previously been proposed to play a role in regulating granulosa cell differentiation and follicle selection. Although AMH receptor type II (AMHR2) dimerizes with a type I receptor to initiate AMH signaling, little is known about the regulation of AMHR2 expression in bovine granulosa cells and the role of AMH in follicle development. The primary objectives of this study were to: (1) characterize AMHR2 expression in granulosa cells during follicle development; (2) identify factors that regulate AMHR2 mRNA expression in granulosa cells; and (3) examine the role of AMH signaling in granulosa cell differentiation and proliferation. Bovine granulosa cells were isolated from 5- to 8-mm follicles before selection and deviation, as well as from 9- to 12-mm and 13- to 24-mm follicles after selection. Analyses revealed that expression of AMHR2 was greater in 5- to 8-mm follicles compared with 13- to 24-mm follicles (P < 0.05). Granulosa cells treated with bone morphogenetic protein 6 (BMP6) or BMP15, but not BMP2, significantly increased AMHR2 expression when compared with control cultured cells (P < 0.05). In addition, expression of AMH was greater in granulosa cells cultured with BMP2, BMP6, or BMP15 when compared with controls (P < 0.05). Finally, treatment with recombinant human AMH, in vitro, inhibited CYP19A1 expression in a dose-related (10-100 ng/mL) fashion, and reduced granulosa cell proliferation at 48 and 72 hours (P < 0.05). Results from these studies indicate that AMH signaling plays a role in both regulating granulosa cell proliferation and preventing granulosa cells from 5- to 8-mm follicles from undergoing premature differentiation before follicle selection.

Characterization of chicken TNFR superfamily decoy receptors, DcR3 and osteoprotegerin.

Tumor necrosis factor (TNF) family ligands bind to death domain-containing TNF receptors (death receptors), which can subsequently activate intracellular signaling pathways to initiate caspase activity and apoptotic cell death. Decoy receptors, without intracellular death domains, have been reported to prevent cytotoxic effects by binding to and sequestering such ligands, or by interfering with death receptor trimerization. The chicken death receptors, Fas, TNFR1, DR6, and TVB, are constitutively expressed in a relatively wide variety of hen tissues. In this study, two chicken receptors with sequence homology to the mammalian decoys, DcR3 and osteoprotegerin, were identified and their pattern of expression was characterized. Unlike the previously identified chicken death receptors, the newly characterized decoy receptors show comparatively limited expression among tissues, suggesting a tissue-specific function. Finally, characterization of these chicken receptors further contributes to understanding the evolutionary divergence of TNFR superfamily members among vertebrate species.

Avian TVB (DR5-like) death receptor expression in hen ovarian follicles.

TVB is an avian death domain-containing receptor belonging to the TNF receptor family and is proposed to be the ortholog to mammalian DR5. Although TVB receptor activation has been demonstrated to mediate apoptosis in chick embryo fibroblasts, there is essentially no information regarding TVB expression or regulation in the mature hen ovary, and in particular within the follicle granulosa layer where apoptosis is known to promote atresia. Significantly, the TVB receptor represents the fourth death domain-containing receptor (also including Fas, TNF-R1, and DR6) found to be expressed within hen granulosa cells. Levels of TVB expression are higher in prehierarchal follicles actively undergoing atresia compared to healthy follicles. However, increased TVB expression does not precede follicle death induced in vitro. Furthermore, TVB expression within granulosa cells is highest during the final stages of follicle development when follicles are not normally susceptible to undergoing atresia. These results provide evidence that TVB receptor signaling in the ovary may function in a capacity other than solely to mediate granulosa cell death and follicle atresia.

Caspase-mediated apoptosis in the vertebrate ovary.

In the vertebrate ovary, apoptosis is the process by which excess or non-viable germ and granulosa cells are eliminated early in ontogeny (often beginning before birth), and thereafter continuously throughout reproductive life. Accordingly, an excessively high rate or abnormal triggering of such cell death (and, by implication, follicle atresia) can negatively affect fertility. Programmed cell death involves the integration of many pathways and intracellular proteins, and central among these at almost every stage are members of the caspase family. Relatively little attention has been focused upon the ovary with regards to elucidating initiator and effector members of the caspase family, and pathways by which they are activated and inactivated. The present review briefly describes vertebrate caspases and the regulation of their function in non-ovarian tissues. Subsequently, the status of caspase expression and function in orchestrating apoptotic cell death in ovarian germ and follicle somatic cells is considered. The most compelling results implicating specific caspases in ovarian function have been derived from mouse single and double knockout model systems. The final outcome of continued studies, in addition to providing information regarding understanding and management of infertility, will influence the development of strategies to treat ovarian cancers and ameliorate the adverse effects of their therapy (for example, chemotherapy).