Regulatory mechanisms of G protein-coupled receptor (gpcr) signaling at follicle selection in the hen ovary

Open Access
Kim, Dongwon
Graduate Program:
Integrative Biosciences
Doctor of Philosophy
Document Type:
Date of Defense:
December 18, 2013
Committee Members:
  • Alan Leslie Johnson, Dissertation Advisor
  • Alan Leslie Johnson, Committee Chair
  • Joy Lee Pate, Committee Member
  • Francisco Javier Diaz, Committee Member
  • Robert Paulson, Special Member
  • ovarian follicle
  • follicle selection
  • granulosa cell
  • granulosa cell differentiation
  • G protein-coupled receptor
  • follicle stimulating hormone receptor
  • vasoactive intestinal peptide receptor
  • beta-arrestin
  • GPCR kinase2
  • desensitization
  • circadian rhythm
  • angiogenesis
  • vascular endothelial growth factor
  • angiopoietin
In vertebrate species that ovulate a species-specific number of eggs each ovulation cycle, ovarian follicles undergo a process of selection for further development and final maturation prior to ovulation. Subsequent to follicle selection, ovarian follicles mature into preovulatory follicles and are eventually ovulated. In monovulatory vertebrate species, which maintain reproductive cycles, only one follicle from the limited growing follicle population is typically selected for fertilization. Ovarian follicle selection is largely dependent upon the transition of granulosa cells (GCs) from an undifferentiated to a fully differentiated state, which accompanies the morphological and functional changes that initiate follicle development. In both mammals and birds, it has been well established that GC differentiation during follicle development is dependent upon gonadotropin-responsiveness (e.g. initially follicle stimulating hormone (FSH)- and subsequently luteinizing hormone (LH)-responsiveness). In the hen ovary, the selection of a single follicle each day occurs from a small cohort of prehierarchal follicles measuring 6-8 mm in diameter. To date, considerable research has been focused on identifying the role of growth factors or the signaling pathways in granulosa cells to understand the principle of follicle selection. Nonetheless, the ultimate signals responsible for cellular events which result in a single follicle being selected have yet to be demonstrated in any vertebrate. The present studies were conducted to investigate the regulatory mechanisms of G protein-coupled receptor (GPCR) signaling to better understand mechanisms that initiate the differentiation of GCs at follicle selection in the hen ovary. It is hypothesized that prior to follicle selection, FSH receptor (FSHR) and vasoactive intestinal peptide (VIP) receptors (VPAC-1 and -2) are desensitized by a βARRESTIN-mediated event in undifferentiated GCs. Additionally, it is proposed that receptor-desensitization is maintained and/or promoted by inhibitory mitogen-activated protein kinase (MAPK) signaling. Therefore, at follicle selection, actively iv differentiating GCs are able to acquire receptor-responsiveness due to reduced MAPK signaling, that results in inducing cyclic 3,5-adenosine monophosphate (cAMP)-induced signaling. Finally, the present studies demonstrate that the acquisition of receptor-responsiveness initiates GC differentiation by promoting cAMP-induced steroidogenesis, clock gene expression, and angiogenesis. Undifferentiated GCs from hen prehierarchal follicles failed to initiate signaling via cyclic 3,5-adenosine monophosphate (cAMP) following a 3-4 h challenge with recombinant human (rh)FSH (10 ng/ml) and chicken (ch)VIP (1 μM), despite the finding that undifferentiated GCs from prehierarchal follicles express FSHR mRNA and FSHR protein plus VPAC1/2 mRNA during follicle development. In addition, these cells demonstrated an inability to induce steroidogenic acute regulatory (STAR) protein expression and progesterone production due to the absence of GPCR-mediated cAMP formation. Specifically, knockdown studies using small interfering RNA (siRNA) specific for Gallus βARRESTIN1 revealed that reduced βARRESTIN expression in undifferentiated GCs results in increased cAMP formation, STAR protein expression, progesterone production following a rhFSH or chVIP treatment compared to cells transfected with non-targeting (scrambled) siRNA. Furthermore, co-transfection of bovine βARRESTIN and GPCR kinase 2 (GRK2) constructs in actively differentiating GCs significantly decreased cAMP and progesterone production following a 3 h rhFSH treatment. In chickens, VIP, unlike the unchanging concentrations of circulating of FSH, is rhythmically expressed within the hypothalamus during a photo-induced reproductive cycle [1, 2]. Accordingly, it was hypothesized that immediately subsequent to follicle selection in the hen, VIP derived from a neuronal and/or humoral origin can serve to regulate expression of clock genes within the GC layer. Significantly, it was determined that cAMP-induced clock gene expression was not initiated by a 4 h challenge with chVIP (1 μM) in undifferentiated GCs. Using βARRESTIN1-siRNA, it was confirmed that undifferentiated GCs with reduced levels of v βARRESTIN became competent to respond to chVIP, which resulted in increasing cAMP-dependent BRAIN AND MUSCLE ARNT-LIKE PROTEIN 1 (BMAL1) gene expression. Finally, BMAL1 and CIRCADIAN LOCOMOTOR OUTPUT CYCLES KAPUT (CLOCK) gene expression in actively differentiating GCs varied in a time-in-culture-dependent fashion, compared to undifferentiated GCs in which clock gene expression demonstrated no rhythmicity. The number and size of blood vessels surrounding and within the follicle theca layer increases dramatically beginning at the time of follicle selection when compared to prehierarchal follicles. However, processes regulating angiogenesis have not been studied in chicken ovarian follicles. There is elevated expression of VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) mRNA in GCs from the selected (preovulatory) follicle, compared to GCs from unselected (prehierarchal) follicles. In addition, it was determined that ANGIOPOIETIN (ANGPT)-2, a natural antagonist for ANGPT-1 with angiogenic functions, is highly expressed in undifferentiated GCs and its gene expression decreases after follicle selection. In hen GCs, it was determined that VEGF and ANGPT-2 gene expression is regulated by gonadotropins and several transforming growth factor beta (TGFβ) superfamily members, including TGFβ1 and BMP4. Levels of VEGF and VEGF RECEPTOR (VEGFR) mRNA were determined to be dramatically lower in atretic follicles compared to healthy prehierarchal follicles, which suggests VEGF signaling may play a role in maintaining follicle survival. Collectively, the present studies provide novel evidence that prior to follicle selection FSHR and VPACs in undifferentiated hen GCs are, at least in part, desensitized by a βARRESTIN-mediated event. This receptor desensitization contributes to the absence of cAMP-mediated signaling, that results in the inability to synthesize steroids, the arrhythmicity of peripheral clock genes, and a minimal extent of vasculature within prehierarchal follicles.