Activation of Latent Human GDF9 by a Single Residue Change (Gly391Arg) in the Mature Domain

Abstract

Growth differentiation factor 9 (GDF9) controls granulosa cell growth and differentiation during early ovarian folliculogenesis and regulates cumulus cell function and ovulation rate in the later stages of this process. Similar to other TGF-β superfamily ligands, GDF9 is secreted from the oocyte in a noncovalent complex with its prodomain. In this study, we show that prodomain interactions differentially regulate the activity of GDF9 across species, such that murine (m) GDF9 is secreted in an active form, whereas human (h) GDF9 is latent. To understand this distinction, we used site-directed mutagenesis to introduce nonconserved mGDF9 residues into the pro- and mature domains of hGDF9. Activity-based screens of the resultant mutants indicated that a single mature domain residue (Gly(391)) confers latency to hGDF9. Gly(391) forms part of the type I receptor binding site on hGDF9, and this residue is present in all species except mouse, rat, hamster, galago, and possum, in which it is substituted with an arginine. In an adrenocortical cell luciferase assay, hGDF9 (Gly(391)Arg) had similar activity to mGDF9 (EC(50) 55 ng/ml vs. 28 ng/ml, respectively), whereas wild-type hGDF9 was inactive. hGDF9 (Gly(391)Arg) was also a potent stimulator of murine granulosa cell proliferation (EC(50) 52 ng/ml). An arginine at position 391 increases the affinity of GDF9 for its signaling receptors, enabling it to be secreted in an active form. This important species difference in the activation status of GDF9 may contribute to the variation observed in follicular development, ovulation rate, and fecundity between mammals.

Effects of species differences on oocyte regulation of granulosa cell function

“…This interpretation is consistent with our hypothesis, namely, that there is no effect of BMP15 immuno-neutralisation on thymidine incorporation by rat or sheep GC co-incubated with rat DO due to species of origin of the growth factors (McNatty et al 2005b). It is also consistent with the recent report by Simpson et al (2012) which showed that there are species differences in the bioactivity of GDF9 due to different amino acid residues at key positions with respect to receptor binding domains. For example, through site-specific mutagenesis, they report that the Gly 391 residue in the mature domain in human GDF9 confers latency whereas in mouse and rat GDF9, the 391 residue is Arg instead of Gly, which presents GDF9 in an active form.…”

“…Furthermore, both the ovine oocyte-secreted factors GDF9 and BMP15 were essential to stimulate 3 H-thymidine incorporation in rat GC. These species differences are likely to be due to both the levels of protein secreted and the bioactivity of the different protein forms (McNatty et al 2005a, 2005b, Simpson et al 2012.…”

Using sheep lines with mutations in single genes to better understand ovarian function

“…New data are emerging regarding the effects of post-translational modifications on the activity of GDF9 and BMP15 (McMahon et al 2008) and the role of sequence in the proregion in regulating activity of the mature region. These may operate via control of processing of the protein and regulating latency of the mature protein or potentially direct actions (McIntosh et al 2008, Tibaldi et al 2010, Simpson et al 2012, Al-Musawi et al 2013. The relative importance of the effects of GDF9 and BMP15 on mural vs cumulus granulosa cell function is also unclear, and sometimes debated.…”

Cumulin, an Oocyte-secreted Heterodimer of the Transforming Growth Factor-β Family, Is a Potent Activator of Granulosa Cells and Improves Oocyte Quality

“…As with all members of the TGF-␤ family, GDF9 and BMP15 are produced as pro-proteins, consisting of an N-terminal proregion and a receptor binding C-terminal mature region. Activation of these factors requires proteolytic processing by a furin-like protease (3,4). For most TGF-␤ mem-bers, the processed mature domains signal as covalently linked homodimers, although covalent heterodimers are also known (inhibin A and B (5)).…”

“…Notably, human GDF9 is latent, because it remains associated with its prodomain, whereas mouse GDF9 is particularly potent, because of its prodomain being readily displaced (3,12,13). By contrast, human BMP15 is recombinantly produced in an active form in mammalian cell culture, whereas mouse BMP15 is not expressed under the same conditions (4).…”