Aberrant GDF9 expression and activation are associated with common human ovarian disorders.
BACKGROUND
Growth differentiation factor 9 (GDF9) is a central regulator of folliculogenesis and ovulation rate. Fourteen mutations in human (h) GDF9 have been reported in women with premature ovarian failure or polycystic ovarian syndrome as well as in mothers of dizygotic twins, implicating GDF9 in the etiology of these conditions. We sought to determine how these mutations alter the biological activity of hGDF9.


OBJECTIVE
The objective of the study was to determine whether aberrant GDF9 expression or activation is associated with common ovarian disorders.


METHODS
Homology modeling was used to predict the location of individual mutations within structurally important regions of the pro domains and mature domains of hGDF9. Each hGDF9 variant was generated by site-directed mutagenesis, expressed from human embryonic kidney 293T cells and assessed as to whether it resulted in defective production or the enhanced activation of mature hGDF9 in an in vitro granulosa cell proliferation bioassay.


RESULTS
Mutations observed in mothers of dizygotic twins (P103S and P374L) completely abrogated GDF9 expression, suggesting that women heterozygous for these mutations would have a 50% reduction in GDF9 levels. Comparable declines in GDF9 in ewes result in a 2-fold increase in ovulation rate and fecundity. Remarkably, three prodomain mutations associated with premature ovarian failure (S186Y, V216M, and T238A) all resulted in the activation of hGDF9. Mechanistically, these mutations reduced the affinity of the prodomain for mature hGDF9, allowing the growth factor to more readily access its signaling receptors.


CONCLUSIONS
Together these findings indicate that alterations to hGDF9 synthesis and activity can contribute to the most common ovarian pathologies.
Prince Henry's Institute of Medical Research (C.M.S., D.M.R., S.L.A.-M., C.A.H., P.G.S.), Clayton, Victoria 3168, Australia; Department of Biochemistry and Molecular Biology (C.M.S., C.A.H., P.G.S.), Monash University, Clayton, Victoria 3800, Australia; School of Biological Sciences (D.A.H., K.P.M.), Victoria University of Wellington, Wellington 6140, New Zealand; and Robinson Institute (L.J.R., D.G.M., R.B.G.), Research Centre for Reproductive Health, School of Paediatrics and Reproductive Health, University of Adelaide, South Australia 5005, Australia.
Courtney M
CM
Simpson
David M
DM
Robertson
Sara L
SL
Al-Musawi
Derek A
DA
Heath
Kenneth P
KP
McNatty
Lesley J
LJ
Ritter
David G
DG
Mottershead
Robert B
RB
Gilchrist
Craig A
CA
Harrison
Peter G
PG
Stanton
D000595:Amino Acid Sequence; D000818:Animals; D016022:Case-Control Studies; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation; D055429:Growth Differentiation Factor 9 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D010049:Ovarian Diseases / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics
C525897:GDF9 protein, human; D055429:Growth Differentiation Factor 9; D050505:Mutant Proteins