title: Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells.
abstract: BACKGROUND
  We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin β(B)-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A.
  
  
  METHODS
  hGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin β(B)-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey's test.
  
  
  RESULTS
  GDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin β(B)-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin β(B)-subunit mRNA and inhibin B production were attenuated by adding FST.
  
  
  CONCLUSIONS
  GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin β(B)-subunit mRNA expression and inhibin B production in hGL cells.
authors.affiliation: Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Feng-Tao
authors.initials: FT
authors.lastname: Shi
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Cheung
authors.forename: He-Feng
authors.initials: HF
authors.lastname: Huang
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Leung
mesh_terms: D000704:Analysis of Variance; D001483:Base Sequence; D017931:DNA Primers; D005260:Female; D038681:Follistatin / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000235:genetics; D038702:Follistatin-Related Proteins / Q000096:biosynthesis* / Q000235:genetics; D055785:Gene Knockdown Techniques; D006107:Granulosa Cells / Q000378:metabolism*; D055429:Growth Differentiation Factor 9 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D014975:Lutein / Q000378:metabolism*; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D038681:Follistatin; D038702:Follistatin-Related Proteins; D055429:Growth Differentiation Factor 9; D014975:Lutein