The
TGF beta family member growth differentiation factor-9 (GDF-9) is an oocyte-derived factor that is essential for mammalian ovarian folliculogenesis.
GDF-9 mRNAs have been shown to be expressed in the human ovarian follicle from the primary follicle stage onward, and recombinant
GDF-9 has been shown to promote human ovarian follicle growth in vitro. In this study with primary cultures of human granulosa-luteal (hGL) cells, we investigated whether recombinant
GDF-9 activates components of the Smad signaling pathways known to be differentially activated by
TGF beta and the
bone morphogenetic proteins (BMPs). As with
TGF beta,
GDF-9 treatment caused the phosphorylation of endogenous 53-kDa
proteins detected in Western blots with antiphospho-Smad2
antibodies (alpha PS2). However, unlike BMP-2,
GDF-9 did not activate the phosphorylation of antiphospho-Smad1 antibody (alphaPS1)-immunoreactive
proteins in hGL cells.
Infection of hGL cells with an adenovirus expressing Smad2 (Ad-Smad2) confirmed that
GDF-9 activates specifically phosphorylation of the
Smad2 protein.
Infection of hGL cells with Ad-Smad7, which expresses the inhibitory
Smad7 protein, suppressed the levels of both GDF-9-induced endogenous and adenoviral alpha PS2-reactive
proteins. Furthermore,
GDF-9 increased the steady state levels of
inhibin beta(B)-subunit mRNAs in hGL cells and strongly stimulated the secretion of dimeric
inhibin B. Again, Ad-Smad7 blocked GDF-9-stimulated
inhibin B production in a concentration-dependent manner. We identify here for the first time distinct molecular components of the
GDF-9 signaling pathway in the human ovary. Our data suggest that
GDF-9 mediates its effect through the pathway commonly activated by
TGF beta and
activin, but not that activated by many BMPs. The results are also consistent with the suggestion that in addition to endocrine control of
inhibin production by
gonadotropins, a local paracrine control of
inhibin production is likely to occur via oocyte-derived factors in the human ovary.