title: Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells.
abstract: Growth differentiation factor-9 (GDF9) is an oocyte secreted paracrine factor essential for mammalian ovarian folliculogenesis. Like other members of the transforming growth factor-beta (TGFbeta) superfamily, GDF9 is synthesized as a prepropeptide which needs processing by furin-like proteases to result in an active mature protein. We have previously characterized a preparation of unpurified recombinant mouse GDF9 which is bioactive as produced by human embryonic kidney 293T (HEK-293T) cells. However, we find that unpurified recombinant human GDF9 (hGDF9) produced by HEK-293T cells is not bioactive. Purified recombinant hGDF9 is bioactive and here we report the characterization of this protein. We find that the purified untagged mature region of hGDF9 is active in transcriptional reporter assays specific for Smad3/4 in human granulosa-luteal (hGL) cells. We also demonstrate the use of a BMP (Smad1/5) responsive (BRE-luciferase) adenovirus in primary cultures of hGL cells to detect BMP responses. Using this adenovirus we find that purified human GDF9 does not activate the Smad1/5 pathway. Purified hGDF9 mature region activated the Smad3 pathway also in the FSH responsive human granulosa tumor cell line KGN. Primary cultures of rat granulosa cells responded to purified hGDF9 with an increase in DNA synthesis as measured by [3H]-thymidine uptake. Here we also report that the inclusion of a C-terminal affinity purification tag destroys GDF9 bioactivity. This study is the first characterization of purified biologically active human GDF9 and as such is of importance for studies on human fertility, and efforts aimed at treating infertility conditions.
authors.affiliation: Department of Bacteriology and Immunology, Haartman Institute, PO Box 21 Haartmaninkatu 3, University of Helsinki, FIN-00014 Helsinki, Finland. David.Mottershead@helsinki.fi
authors.forename: David G
authors.initials: DG
authors.lastname: Mottershead
authors.forename: Minna M
authors.initials: MM
authors.lastname: Pulkki
authors.forename: Pranuthi
authors.initials: P
authors.lastname: Muggalla
authors.forename: Arja
authors.initials: A
authors.lastname: Pasternack
authors.forename: Minna
authors.initials: M
authors.lastname: Tolonen
authors.forename: Samu
authors.initials: S
authors.lastname: Myllymaa
authors.forename: Olexandr
authors.initials: O
authors.lastname: Korchynskyi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nishi
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Yanase
authors.forename: Stan
authors.initials: S
authors.lastname: Lun
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Juengel
authors.forename: Mika
authors.initials: M
authors.lastname: Laitinen
authors.forename: Olli
authors.initials: O
authors.lastname: Ritvos
mesh_terms: D000818:Animals; D055430:Bone Morphogenetic Protein 15; D002460:Cell Line; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism*; D055429:Growth Differentiation Factor 9; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism; D013936:Thymidine; D014316:Tritium
chemical_list: C525901:BMP15 protein, human; C525902:Bmp15 protein, mouse; C525904:Bmp15 protein, rat; D055430:Bone Morphogenetic Protein 15; D017077:Culture Media, Conditioned; C525897:GDF9 protein, human; C525898:Gdf9 protein, mouse; C525899:Gdf9 protein, rat; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D051785:Smad Proteins; D014316:Tritium; D013936:Thymidine