Poster Presentation The Annual Scientific Meeting of the Endocrine Society of Australia and the Society for Reproductive Biology 2014

Oct4-GFP expression during transformation of gonocytes into spermatogonial stem cells in the perinatal mouse testis (#353)

Ruili Li 1 2 , Amanda Vannitamby 1 , Jian-Guo Zhang 3 , Emma L Fehmel 4 , Bridget R Southwell 1 2 , John M Hutson 1 2 5
  1. F. Douglas Stephens Surgical Research Laboratory, Murdoch Children"s Research Institute, Parkville,, Victoria, Australia
  2. Department of Paediatrics, University of Melbourne, Parkville,, Victoria, Australia
  3. Walter and Eliza Hall Institute of Medical Research, , Parkville,, Victoria, Australia
  4. Central Medical School, Faculty of Medicine, Nursing and Health science, Monash University, Clayton, Victoria, Australia
  5. Urology Department, Royal Children’s Hospital, Parkville,, Victoria, Australia

Human testes normally descend before birth. However, 2-5% of boys are born with undescended testis (UDT), 30-60% of them may be subsequently infertile, with 3-5 times higher risk of developing germ cell (GC) tumour than boys with descended testes [1]. We hypothesize that the risk of infertility or tumour with UDT is related to impaired transformation of gonocytes into spermatogonial stem cells (SSC). Our previous study suggested that gonocytes transform into SSC during migration from the centre of testicular tubule to the basement membrane (BM) [2]. In this study we examined gonocyte transformation in perinatal testes using established Oct4 promoter-driving EGFP (OG2) mice.

Material and Methods
Testes were collected from E17 (day of vaginal plug = E0) fetuses and postnatal (P) day P0 (birth), P2, P4, P6, P8 and P10 pups (OG2 x CD1 mice), fixed in 4% PFA for immunohistochemistry or frozen to -80°C for immunoblotting. Immunofluorescent confocal images were captured for presence of MVH, AMH, NGN3, MT1-MMP, C-kit and Ki67 in OCT-embedded tissue sections. GFP+, GFP- germ cells in cross-sectioned testicular tubules in confocal images were counted and analysed. Testicular proteins were analyzed by immunoblotting.

All gonocytes at E17 and P0 were Oct4-GFP+ located centrally in testicular cords/tubules and were Ki67-. Gonocytes became Ki67+ and began migrating to BM at P2. Majority gonocytes migrated to the BM and remained GFP+/cKit-/Ki67+ by P4 with some GFP+/c-Kit+/Ki67+ GC. Oct4-GFP- /c-Kit+ GC appeared at P6 and increased significantly by P10. NGN3 and MT1-MMP were strongly expressed in E17 gonocytes, then decreased postnatally when gonocytes migrated towards BM.
In conclusion, our study indicates that gonocyte (Oct4-GFP+/c-Kit-/Ki67-) transformation starts postnatally with migration to BM to become prospermatogonia (Oct4-GFP+/c-Kit-/Ki67+) by P4. These germ cells further differentiate into type A spermatogonia (Oct4-GFP+/c-Kit+/Ki67+) from P4 to P10 at BM.

  1. Hutson, J.M., et al., Germ cell development in the postnatal testis: the key to prevent malignancy in cryptorchidism? Front Endocrinol (Lausanne), 2012. 3: p. 176.
  2. Li, R., et al., Is matrix metalloproteinase required in postnatal testicular tubules for germ cell maturation? J Pediatr Surg, 2012. 47(9): p. 1724-9.