title: Exogenous Secreted Frizzled-Related Protein-4 Modulates Steroidogenesis of Rat Granulosa Cells through Wnt/β-catenin and PI3K/AKT Signaling Pathways.
abstract: BACKGROUND
  It has been reported that secreted frizzled-related protein-4 known as an antagonist of Wnt signaling pathway plays a role in luteinization process of rodent granulosa cells. The purpose of this study was twofold: 1) to determine whether recombinant human secreted frizzled-related protein-4 (rhSFRP-4) could directly induce terminal differentiation of rat Granulosa Cells (GCs) and 2) to understand how the modulation of β-catenin and Protein Kinase B (PKB)/AKT activity by exogenous SFRP-4 could be involved in steroidogenesis.
  
  
  METHODS
  GCs were firstly stimulated with Follicle-Stimulating Hormone (FSH) named as FSH-primed cells then were treated with luteinizing hormone (LH). Then estradiol (E2) and progesterone (P4) production levels were assessed in the absence or presence of rhSFRP-4 treatment. The expression levels of activated β-catenin, pAKTser473, pGSK3βser9were assessed by western blot or immunofluoresence.
  
  
  RESULTS
  In the presence of rhSFRP-4, there was 38% decreased E2levels compared to untreated FSH-primed cells (p<0.05), and P4production subsequently decreased. However, in GCs pre-treated with rhSFRP-4 prior to addition of FSH, P4levels increased 2-fold compared with untreated cells (p<0.05). Unexpectedly, treatment with rhSFRP-4 prior to LH stimulation inhibited LH-induced P4secretion. Treatment with low (0.5ng/ml) but not high (50ng/ml) concentrations of rhSFRP-4 led to significantly increased levels of pGSK3βser9(1.6-fold) and nuclear active β-catenin (2.8-fold) in GCs compared with untreated cells. Interestingly, pre-treating GCs with rhsFPR4 prior to LH stimulation resulted in a 38% decrease in pAKTser473levels compared with those in LH-treated cells (p<0.05).
  
  
  CONCLUSIONS
  Taken together, our results showed that rhSFRP-4 could directly induce terminal differentiation in GCs via the modulation of β-catenin and PKB/AKT pathways and that it does so in a dose-dependent manner.
authors.affiliation: Department of Animal Physiology, Developmental Biology Laboratory, Faculty of Biology, University College of Science, University of Tehran, Tehran, Iran.
authors.forename: Ghamartaj
authors.initials: G
authors.lastname: Hossein
authors.affiliation: Department of Animal Physiology, Developmental Biology Laboratory, Faculty of Biology, University College of Science, University of Tehran, Tehran, Iran; Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
authors.forename: Manijeh
authors.initials: M
authors.lastname: Khanmohammadi
authors.affiliation: Department of Animal Physiology, Developmental Biology Laboratory, Faculty of Biology, University College of Science, University of Tehran, Tehran, Iran; Department of Stem Cells & Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology & Technology, ACECR, Tehran, Iran.
authors.forename: Parisa
authors.initials: P
authors.lastname: Sahranavard Fard
authors.affiliation: Department of Animal Physiology, Developmental Biology Laboratory, Faculty of Biology, University College of Science, University of Tehran, Tehran, Iran.
authors.forename: Yasaman
authors.initials: Y
authors.lastname: Heidarian
authors.affiliation: Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
authors.forename: Somaieh
authors.initials: S
authors.lastname: Kazemnejad
authors.affiliation: Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
authors.forename: Mohammad Mehdi
authors.initials: MM
authors.lastname: Akhondi
keywords: Active β-catenin; GSK3β; PKB/AKT; Rat granulosa cell; Secreted frizzled-related protein-4 (SFRP-4)