title: Growth differentiation factor-9 stimulates inhibin production and activates Smad2 in cultured rat granulosa cells.
abstract: Ovarian inhibin production is stimulated by FSH and several TGFbeta family ligands including activins and bone morphogenetic proteins. Growth differentiation factor-9 (GDF-9) derived by the oocyte is a member of the TGFbeta/activin family, and we have previously shown that GDF-9 treatment stimulates ovarian inhibin-alpha content in explants of neonatal ovaries. However, little is known about GDF-9 regulation of inhibin production in granulosa cells and downstream signaling proteins activated by GDF-9. Here, we used cultured rat granulosa cells to examine the influence of GDF-9 on basal and FSH-stimulated inhibin production, expression of inhibin subunit transcripts, and the GDF-9 activation of Smad phosphorylation. Granulosa cells from small antral follicles of diethylstilbestrol-primed immature rats were cultured with FSH in the presence or absence of increasing concentrations of GDF-9. Secreted dimeric inhibin A and inhibin B were quantified using specific ELISAs, whereas inhibin subunit RNAs were analyzed by Northern blotting using (32)P-labeled inhibin subunit cDNA probes. Similar to FSH, treatment with GDF-9 stimulated dose- and time-dependent increases of both inhibin A and inhibin B production. Furthermore, coincubation of cells with GDF-9 and FSH led to a synergistic stimulation of both inhibin A and inhibin B production. GDF-9 treatment also increased mRNA expression for inhibin-alpha and inhibin-beta subunits. To investigate Smad activation, granulosa cell lysates were analyzed in immunoblots using antiphosphoSmad1 and antiphosphoSmad2 antibodies. GDF-9 treatment increased Smad2, but not Smad1, phosphorylation with increasing doses of GDF-9 leading to a dose-dependent increase in phosphoSmad2 levels. To further investigate inhibin-alpha gene promoter activation by GDF-9, granulosa cells were transiently transfected with an inhibin-alpha promoter-luciferase reporter construct and cultured with different hormones before assaying for luciferase activity. Treatment with FSH or GDF-9 resulted in increased inhibin-alpha gene promoter activity, and combined treatment with both led to synergistic increases. The present data demonstrate that oocyte-derived GDF-9, alone or together with pituitary-derived FSH, stimulates inhibin production, inhibin subunit mRNA expression, and inhibin-alpha promoter activity by rat granulosa cells. The synergistic stimulation of inhibin secretion by the paracrine hormone GDF-9 and the endocrine hormone FSH could play an important role in the feedback regulation of FSH release, thus leading to the modulation of follicle maturation and ovulation.
authors.affiliation: Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317, USA.
authors.forename: Jae-Sook
authors.initials: JS
authors.lastname: Roh
authors.forename: Jonas
authors.initials: J
authors.lastname: Bondestam
authors.forename: Sabine
authors.initials: S
authors.lastname: Mazerbourg
authors.forename: Noora
authors.initials: N
authors.lastname: Kaivo-Oja
authors.forename: Nigel
authors.initials: N
authors.lastname: Groome
authors.forename: Olli
authors.initials: O
authors.lastname: Ritvos
authors.forename: Aaron J W
authors.initials: AJ
authors.lastname: Hsueh
mesh_terms: D000818:Animals; D055430:Bone Morphogenetic Protein 15; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004357:Drug Synergism; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D055429:Growth Differentiation Factor 9; D007265:Inhibins / Q000096:biosynthesis* / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007700:Kinetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D051899:Smad2 Protein; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: C525904:Bmp15 protein, rat; D055430:Bone Morphogenetic Protein 15; D004268:DNA-Binding Proteins; C525899:Gdf9 protein, rat; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D015534:Trans-Activators; C422362:inhibin A; C422364:inhibin B; C094377:inhibin-alpha subunit; D007265:Inhibins; D005640:Follicle Stimulating Hormone