Oral Presentation ESA-SRB Conference 2015

Delineation of myeloid lineage derived immune cells in the adult mouse testis (#29)

Sivanjah Indumathy 1 2 , Britta Klein 3 , Hans-christian Schuppe 4 , Martin Bergmann 3 , Nicolas Da Silva 5 , Michael Hickey 2 , Mark Hedger 1 , Kate Loveland 2
  1. Centre for Reproductive Health, The Hudson Institute of Medical Research, Clayton, Victoria, Australia
  2. School Clinical Sciences, Monash University, Clayton, Victoria, Australia
  3. Department of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University, Giessen, Germany
  4. Clinic of Urology, Pediatric Urology and Andrology, Justus-Liebig-University, Giessen, Germany
  5. Massachusetts General Hospital (MGH) and Harvard Medical School , Boston, Massachusetts, United States

The ability of the testis to tolerate spermatogenic cell autoantigens is crucial for sperm production and any compromise can lead to male infertility. Although the testicular immune cells in the adult have been studied in detail [1-3], the developmental dynamics of these populations have yet to be characterised. Macrophages are commonly studied in inflammatory diseases; their role in organogenesis is an emerging topic, particularly relating to testis. In adult testes, macrophages closely surround Leydig cells, suggesting they communicate directly [1].  Ethane dimethane sulphonate-induced loss of Leydig cells caused macrophage depletion, and macrophage recovery occurred only after Leydig cell repopulation, highlighting the interdependence of these two cell types [1]. Macrophages have also recently been implicated in fetal testis cord morphogenesis and vascularization [2]. We hypothesised that postnatal testis development is also associated with significant changes in myeloid-derived leukocytes. To address this, we are using immunofluorescence to visualise the localisation of testicular macrophages and dendritic cells within adult testes of transgenic CX3CR1-GFP (macrophage) and CD11c-YFP (dendritic cell) mouse models. In addition, flow cytometry experiments will quantify these cells using interstitial cells collected from mechanically dissociated adult mouse testes. Antibodies [CD45 (leukocytes), F4/80 (macrophages) and CD11c (dendritic cells)] were used to identify immune cell populations. Matched isotype controls in a fluorescence minus one setup and single stain controls enabled optimization for flow cytometry parameters and post-analysis gating of leukocytes. Samples were analysed using a BD LSRII flow cytometer. Approximately 2.5-3% of testicular cells isolated were leukocytes with macrophages accounting for the majority (2-2.5% of total). Furthermore, a unique population co-expressing CD11c+F4/80+ was identified for the first time in the testes. A combination of immunofluorescence and flow cytometry studies using transgenic mouse models will reveal the dynamic composition of testicular immune cells and their possible contribution to organ development and maintenance.

  1. Winnall, W.R., J.A. Muir, and M.P. Hedger, Rat resident testicular macrophages have an alternatively activated phenotype and constitutively produce interleukin-10 in vitro. J Leukoc Biol, 2011. 90(1): p. 133-43.
  2. Tompkins, A.B., et al., Characterization of lymphocytes in the adult rat testis by flow cytometry: effects of activin and transforming growth factor beta on lymphocyte subsets in vitro. Biol Reprod, 1998. 58(4): p. 943-51.
  3. Wang, J., et al., Leukocyte populations of the adult rat testis following removal of the Leydig cells by treatment with ethane dimethane sulfonate and subcutaneous testosterone implants. Biol Reprod, 1994. 51(3): p. 551-61.
  4. Miller, S.C., B.M. Bowman, and H.G. Rowland, Structure, cytochemistry, endocytic activity, and immunoglobulin (Fc) receptors of rat testicular interstitial-tissue macrophages. Am J Anat, 1983. 168(1): p. 1-13.
  5. DeFalco, T., et al., Yolk-sac-derived macrophages regulate fetal testis vascularization and morphogenesis. Proc Natl Acad Sci U S A, 2014. 111(23): p. E2384-93.