Recombinant growth differentiation factor-9 (GDF-9) enhances growth and differentiation of cultured early ovarian follicles

Transgenic mice with deletion of the GDF-9 (growth differentiation factor-9 ) gene are characterized by the arrest of ovarian follicle development at t he primary stage. Based on the hypothesis that GDF-9 is important for early follicle development, we isolated rat GDF-9 complementary DNA (cDNA) and g enerated recombinant GDF-9 protein to study its physiological role. Using b acteria-derived GDF-9-glutathione S-transferase (GST) fusion protein, speci fic antibodies to the mature form of GDF-9 was generated. Immunohistochemic al staining of ovarian sections indicated the localization of GDF-9 protein in the oocyte of primary, secondary and preantral follicles, whereas immun oblotting demonstrated the secretion of GDF-9 by mammalian cells transfecte d with GDF-9 cDNAs. Recombinant GDF-9 was shown to be an N-glycosylated pro tein capable of stimulating early follicle development. Growth of preantral follicles isolated from immature rats was enhanced by treatment with eithe r GDF-9 or FSH whereas the combined treatment showed an additive effect. In addition, treatment with GDF-9, like forskolin, also stimulated inhibin-ol content in explants of neonatal ovaries. In contrast, the stimulatory effe cts of GDF-9 were not mimicked by amino-terminal tagged GDF-9 that was appa rently not bioactive. Thus, the present study demonstrates the important ro le of GDF-9 in early follicle growth and differentiation. The availability of recombinant bioactive GDF-9 allows future studies on the physiological r ole of GDF-9 in ovarian development in vivo.