Oral Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

Delineating the macrophage population during the first wave of mouse spermatogenesis (#51)

Sivanjah Indumathy 1 2 3 , Britta Klein 4 , Hans-Christian Schuppe 5 , Martin Bergmann 4 , Nicolas Da Silva 6 , Michael Hickey 3 , Mark Hedger 1 , Kate Loveland 1 2 3
  1. Centre for Reproductive Health, The Hudson Institute of Medical Research, Clayton, Victoria, Australia
  2. Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia
  3. School Clinical Sciences, Monash University, Clayton, Victoria, Australia
  4. Department of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University, Giessen, Germany
  5. Clinic of Urology, Pediatric Urology and Andrology, Justus-Liebig-University, Giessen, Germany
  6. Massachusetts General Hospital (MGH) and Harvard Medical School , Boston, Massachusetts, United States

The testis is an immunologically privileged site. Maintenance of an immunosuppressive environment is crucial for sperm production and its impairment can lead to male infertility. Macrophages have recently been implicated in cord morphogenesis and vascularisation in the fetal murine testis, however knowledge of this leukocyte population during the first wave of spermatogenesis is limited. We hypothesized that postnatal testis development is accompanied by a substantial change in macrophage subpopulations. To address this, the CX3CR1-GFP mouse model was examined to identify macrophages using fluorescence. Testes fixed in 4% paraformaldehyde were processed through sucrose gradients and snap-frozen in OCT. Whole testes were fully sectioned (25-100 μm), and a section from each of 3 regions (top, middle, bottom) stained with DAPI for imaging with a Leica SP5 confocal microscope. CX3CR1-GFP+ cells were manually counted using Fiji with Cell Counter and Grid plugins. GFP+ cells were categorised based on morphology and localisation. Macrophages (CX3CR1-GFP+) are present within the interstitium at 0-2, 20-25 and 50-60 days post-partum (dpp). Preliminary analysis revealed a substantial increase in GFP+ macrophages between at 0-2 and 20-25 dpp. In addition, two macrophage phenotypes are readily distinguished at 20-25 and 50-60 dpp. In one, stellate/dendriform cells are widely spaced around the outer tubule surface, while the other appears in interstitial clusters. Flow cytometric analysis of adult testes revealed the leukocyte population consists predominantly of macrophages (70%) and is comprised of heterogeneous subpopulations expressing surface markers for CX3CR1 (fractalkine receptor), F4/80 and CD11c. Phenotypic variants amongst macrophages may reflect functional differences associated with roles in immune surveillance, testis development, or support of spermatogenesis. This study is the first to reveal testicular macrophages in the juvenile mouse testis, providing a basis for understanding their potential contributions, as spermatozoa are first formed and fertility is established.