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-ß (TGFß) 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 (BREluciferase) 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 [3 H]-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.

The bioactivity of human bone morphogenetic protein-15 is sensitive to C-terminal modification: Characterization of the purified untagged processed mature region

“…The usage of the pGL3BRE-luciferase reporter plasmid (Korchynskyi and ten Dijke 2002) and generation of the recombinant adenovirus BRE-Luc has been previously described (Mottershead et al 2008). …”

“…We have previously optimized the usage of a reporter BRE-Luc adenovirus (Mottershead et al 2008) which was generated accordingly to provided protocol using AdEasy system as described previously (He et al 1998). Viruses were amplified and titrated in transcomplemental 293A cells and purified with cesium chloride gradient ultracentrifugation as described previously (He et al 1998).…”

“…The various forms of unpurified BMP15 in the conditioned media were tested for bioactivity via monitoring Smad1/5/8 activation. This was accomplished by transducing human granulosa luteal (hGL) cells with an adenovirus encoding the Smad1/5/8 responsive BRE-luciferase reporter containing BMP Smad binding sites derived from the Id1 promoter (Korchynskyi and ten Dijke 2002;Mottershead et al 2008). Similarly to mouse GDF9 (mGDF9) (Mottershead et al 2008), hBMP15 is produced by HEK-293T cells in an active form, but the bioactivity is lost by Cterminal modification (Fig.…”

“…A preferable situation however, is to be able to purify the protein of interest (in this case hBMP15) without modification by the addition of any epitope tags. We determined in a previous study (Mottershead et al 2008) that the purified untagged mature region of recombinant human GDF9 is biologically active. The approach used in that study was subsequently used to purify rhBMP15 from conditioned media.…”

“…We have recently characterized recombinant human GDF9 (hGDF9) signaling in ovarian granulosa cells and found that the position of the affinity purification tag crucially effects GDF9 bioactivity (Mottershead et al 2008). We found that a C-terminal His 6 tag destroys GDF9 bioactivity, the purified untagged mature region of recombinant human GDF9 being biologically active, exhibiting the ability to activate a Smad3/4 specific transcriptional reporter in human granulosa-luteal cells.…”