The Joint Annual Scientific Meetings of the Endocrine Society of Australia and the Society for Reproductive Biology 2018

Human and mouse FGF9 mutations affect male sex determination (#63)

Brittany Croft 1 2 , Anthony Bird 2 , Makoto Ono 3 , Stefanie Eggers 4 , Stefan Bagheri-Fam 2 , Liang Zhao 5 , Janelle Ryan 2 , Patrick Western 2 , Andrew Kueh 6 , Elizabeth Thompson 7 , Tim Thomas 6 , Peter Stanton 2 , Massayo Harada 8 , Peter Koopman 5 , Andrew Sinclair 4 , Vincent Harley 2
  1. Department of Molecular & Translational Science, Monash University, Melbourne, Victoria, Australia
  2. Hudson Institute of Medical Research, Clayton, VICTORIA, Australia
  3. Department of Paediatrics, Tokyo Bay Urayasu Ichikawa Medical Centre, Chiba, Japan
  4. Murdoch Children's Research Institute , Melbourne, Victoria, Australia
  5. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia
  6. Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
  7. SA Clinical Genetics Service, Women’s and Children’s Hospital, Adelaide, South Australia, Australia
  8. Department of Clinical Anatomy, Tokyo Medical and Dental University, Tokyo, Japan

Disorders of Sex Development (DSDs) encompass a wide spectrum of conditions and often manifest with atypical gonads or genitalia. The majority of 46,XY DSD patients cannot be given an accurate diagnosis, which severely compromises their clinical management.

In the mouse, FGF9 is a key testis-determining gene which functions by repressing pro-ovarian signalling pathways such as WNT4/RSPO1 and FOXL2. This maintains sufficient SOX9 expression in the somatic cells of the embryonic gonad to drive Sertoli cell differentiation and ultimately, male testicular development. Loss of Fgf9 results in complete male to female sex reversal. Yet FGF9 mutants that impair FGF9 homodimerization and FGF receptor binding have only been examined in skeletal defects.

Here we investigate the requirement of FGF9 dimer formation and receptor binding in testicular development, using two homodimer-defective FGF9 mutants from an established mouse model and a human 46,XY DSD patient. 

In humans we have identified a 46,XY Gonadal Dysgenesis DSD patient with an amino acid substitution (D195N), previous studies indicated that the D195 residue is critical for the homodimerization of FGF9. The purified recombinant FGF9D195N protein showed reduced affinity for heparin, a property required for homodimerization. In vitro analysis showed reduced ability to induce Sertoli cell proliferation, which is required for normal testis development.

In mice, we examined the spontaneous mouse Elbow knee synostosis (Eks) which harbours a missense mutation in Fgf9 (N143T), that impairs FGF9 homodimerization. Examination of XY Fgf9N143T/N143T gonads showed delayed testes cord development and ectopic expression of the female Granulosa cell marker FOXL2 at the gonadal poles, indicative of XY sex reversal.

Our results suggest that FGF9 homodimerization and heparin binding are required for FGF9 function during testis development. As a disruption in one or both of these pathways causes sex reversal.