Growth Differentiation Factor-9 Stimulates Inhibin Production and Activates Smad2 in Cultured Rat Granulosa Cells

• Published in: Endocrinology (Philadelphia) 2003-01, Vol.144 (1), p.172-178
• Main Authors: Roh, Jae-Sook, Bondestam, Jonas, Mazerbourg, Sabine, Kaivo-Oja, Noora, Groome, Nigel, Ritvos, Olli, Hsueh, Aaron J. W
• Format: Article
• Language: English
• Subjects: Activation analysis; Activin; Animals; Biological and medical sciences; Bone growth; Bone Morphogenetic Protein 15; Bone morphogenetic proteins; Cell activation; Cell differentiation; Cells, Cultured; Combined treatment; Diethylstilbestrol; Differentiation; DNA-Binding Proteins - metabolism; Drug Synergism; Explants; Female; Follicle Stimulating Hormone - pharmacology; Follicle-stimulating hormone; Follicles; Fundamental and applied biological sciences. Psychology; Gametocytes; Gene expression; Granulosa cells; Granulosa Cells - drug effects; Granulosa Cells - metabolism; Growth Differentiation Factor 9; Hormones; Inhibin; Inhibins - biosynthesis; Inhibins - genetics; Intercellular Signaling Peptides and Proteins - pharmacology; Kinetics; Lysates; Mammalian female genital system; Morphology. Physiology; Neonates; Northern blotting; Ovaries; Ovulation; Paracrine signalling; Phosphorylation; Pituitary; Promoter Regions, Genetic; Proteins; Rats; Rats, Sprague-Dawley; RNA, Messenger - analysis; Smad protein; Smad2 Protein; Stimulation; Trans-Activators - metabolism; Vertebrates: reproduction
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2002-220618

Ovarian inhibin production is stimulated by FSH and several TGFβ family ligands including activins and bone morphogenetic proteins. Growth differentiation factor-9 (GDF-9) derived by the oocyte is a member of the TGFβ/activin family, and we have previously shown that GDF-9 treatment stimulates ovarian inhibin-α 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 32P-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-α and inhibin-β 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-α gene promoter activation by GDF-9, granulosa cells were transiently transfected with an inhibin-α promoter-luciferase reporter construct and cultured with different hormones before assaying for luciferase activity. Treatment with FSH or GDF-9 resulted in increased inhibin-α 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-α 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 regulat