title: Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells.
abstract: In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), bone morphogenetic protein-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1-5 mm) and large (8-22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150-600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P < 0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P < 0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P < 0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P > 0.10). Treatment with 600 ng/ml GDF-9 increased (P < 0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P < 0.05) progesterone production (by 28%), increased (P < 0.05) cell numbers (by 60%), and had no effect (P > 0.10) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13-48%) and estradiol (by 20-51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P < 0.05) steroidogenesis by 27-42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation.
authors.affiliation: Department of Animal Science, Oklahoma State University, Stillwater, Oklahoma 74078, USA. igf1Leo@okstate.edu
authors.forename: L J
authors.initials: LJ
authors.lastname: Spicer
authors.forename: P Y
authors.initials: PY
authors.lastname: Aad
authors.forename: D
authors.initials: D
authors.lastname: Allen
authors.forename: S
authors.initials: S
authors.lastname: Mazerbourg
authors.forename: A J
authors.initials: AJ
authors.lastname: Hsueh
mesh_terms: D000818:Animals; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002417:Cattle; D002452:Cell Count; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D017077:Culture Media, Conditioned; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000096:biosynthesis; D005260:Female; D005640:Follicle Stimulating Hormone / Q000502:physiology*; D006107:Granulosa Cells / Q000187:drug effects / Q000502:physiology*; D055429:Growth Differentiation Factor 9; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D011374:Progesterone / Q000096:biosynthesis; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D051785:Smad Proteins / Q000378:metabolism
chemical_list: D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D017077:Culture Media, Conditioned; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D051785:Smad Proteins; D011374:Progesterone; D004958:Estradiol; D007334:Insulin-Like Growth Factor I; D005640:Follicle Stimulating Hormone