Growth differentiation factor-9 stimulates inhibin production and activates Smad2 in cultured rat granulosa cells

Jaesook Roh, Jonas Bondestam, Sabine Mazerbourg, Noora Kaivo-Oja, Nigel Groome, Olli Ritvos, Aaron J.W. Hsueh

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Abstract

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 antiphospho-Smad1 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 regulation of FSH release, thus leading to the modulation of follicle maturation and ovulation.

Original languageEnglish
Pages (from-to)172-178
Number of pages7
JournalEndocrinology
Volume144
Issue number1
DOIs
StatePublished - 2003 Jan 1

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Growth Differentiation Factor 9
Inhibins
Granulosa Cells
Activins
Hormones
Luciferases
Oocytes
Therapeutics
Phosphorylation

Cite this

Roh, J., Bondestam, J., Mazerbourg, S., Kaivo-Oja, N., Groome, N., Ritvos, O., & Hsueh, A. J. W. (2003). Growth differentiation factor-9 stimulates inhibin production and activates Smad2 in cultured rat granulosa cells. Endocrinology, 144(1), 172-178. https://doi.org/10.1210/en.2002-220618
Roh, Jaesook ; Bondestam, Jonas ; Mazerbourg, Sabine ; Kaivo-Oja, Noora ; Groome, Nigel ; Ritvos, Olli ; Hsueh, Aaron J.W. / Growth differentiation factor-9 stimulates inhibin production and activates Smad2 in cultured rat granulosa cells. In: Endocrinology. 2003 ; Vol. 144, No. 1. pp. 172-178.
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Roh, J, Bondestam, J, Mazerbourg, S, Kaivo-Oja, N, Groome, N, Ritvos, O & Hsueh, AJW 2003, 'Growth differentiation factor-9 stimulates inhibin production and activates Smad2 in cultured rat granulosa cells', Endocrinology, vol. 144, no. 1, pp. 172-178. https://doi.org/10.1210/en.2002-220618

Growth differentiation factor-9 stimulates inhibin production and activates Smad2 in cultured rat granulosa cells. / Roh, Jaesook; Bondestam, Jonas; Mazerbourg, Sabine; Kaivo-Oja, Noora; Groome, Nigel; Ritvos, Olli; Hsueh, Aaron J.W.

In: Endocrinology, Vol. 144, No. 1, 01.01.2003, p. 172-178.

Research output: Contribution to journalArticle

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T1 - Growth differentiation factor-9 stimulates inhibin production and activates Smad2 in cultured rat granulosa cells

AU - Roh, Jaesook

AU - Bondestam, Jonas

AU - Mazerbourg, Sabine

AU - Kaivo-Oja, Noora

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AU - Hsueh, Aaron J.W.

PY - 2003/1/1

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