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

Human multipotent NT2/D1 cells model bipotential events in mammalian sex determination (#298)

Aleisha Symon 1 , Janelle Ryan 1 , Brittany Croft 1 2 , Allara Zylberberg 1 3 , Daniel Bird 1 , Dimuthu Alankarage 4 , Vincent Harley 1
  1. Hudson Institute of Medical Research, Melbourne, VIC, Australia
  2. Murdoch Children's Research Institute, Melbourne, VIC, Australia
  3. Department of Anatomy and Developmental Biology, Monash University, Melbourne, VIC, Australia
  4. Victor Chang Cardiac Research Institute, Sydney, NSW, Australia

Sex determination is the process during embryogenesis when the bipotential gonads differentiate into either testes in males or ovaries in females. In males, the Y chromosome gene SRY turns up SOX9 transcription, directing the somatic cells to differentiate into testicular Sertoli cells rather than ovarian granulosa cells. NT2/D1 cells are a human Sertoli-like cell line derived from a testicular seminoma. Despite being multipotent, they express important Sertoli cell markers at low levels that can be manipulated to recapitulate key male sex determination processes. For example, transfection of SRY turns up SOX9 transcription. SRY and SOX9 play key roles in male sex development since human SRY and SOX9 mutations cause Disorders of Sex Development (DSD) in XX males (SOX9 duplications) and XY females (SOX9 mutations/deletions). However many causes of DSD remain unexplained genetically.

Using the NT2/D1 cells, we are unravelling the role of genes and their relevance to human sex determination and to DSD. We reasoned that the genes regulated by SOX9 are potential DSD genes. Previously we identified 119 candidate SOX9 target genes, including Nedd9, by integrating RNAseq analysis on mouse Sox9 knock-out gonads with Sox9 ChIPseq on wildtype mouse testes (Rahmoun et al., 2017, NAR). Using NT2/D1 cells, overexpression and knockdown of SOX9 led to an increase or decrease in NEDD9 mRNA and protein levels. This suggested that human NEDD9 is a target gene of SOX9. As such, Nedd9 may have an important role to play in testis development. Nedd9 knockout mouse testes show growth defects and a coelomic blood vessel defect. Nedd9 is a scaffolding protein from the Cas family, and using NT2 cells we are currently investigating its role in cellular processes including cell polarity, migration, adhesion, apoptosis and cell cycle control.