Oral Presentation ESA-SRB-APEG-NZSE 2022

Activin A levels regulate fetal testicular macrophages in mice (#29)

Samira Hosseini 1 2 , Daniela Prof Fietz 3 , Penny Whiley 1 4 , Mark Prof Hedger 1 2 5 , Hans-Christian Prof Schuppe 4 , Kate Prof Loveland 1 2 5
  1. Department of Molecular and Translational Sciences, Centre for Reproductive Health , Monash University, Clayton, VIC, Australia
  2. Hudson Institute of Medical Research School of Clinical Sciences, Monash University, Clayton, VIC, Australia
  3. Institute of Veterinary Anatomy, Histology and Embryology, Justus-Liebig University Giessen, Giessen, Germany
  4. Department of Urology, Paediatric Urology and Andrology, Justus Liebig University Giessen, Giessen, Germany
  5. Department of Anatomy and Developmental Biology, School of Biomedical Sciences , Monash University, Clayton, VIC, Australia

There is accumulating evidence that immune cells serve central roles in testis development. Our recent work delineated immune cell emergence in human and murine fetal testes documented innate immune cells' early arrival and predominance (Hosseini et al. 2022, and unpublished). In human pregnancies, activin A levels can be altered in conditions such as maternal infection, preeclampsia and pharmaceutical ingestion, and this could significantly impact macrophage function and polarization towards pro-inflammatory or anti-inflammatory phenotypes. This study investigates whether activin A levels influence the establishment of fetal testicular macrophages in the mouse by interrogating their number, distribution and phenotype. Whole testes were collected from two knockout (KO) mouse models, Inhba (lacking activin A) and Inha (high activin), with wildtype (WT) littermates at embryonic day (E)13.5, E15.5, and postnatal day 0 (PND0). Samples were fixed in 4% paraformaldehyde and paraffin-embedded for immunohistochemistry (IHC F4/80, to detect macrophages; n=4-8 per age per genotype) and snap-frozen to measure transcripts encoding 33 proteins found in immune cells using Fluidigm qRT-PCR (n=3/age genotype). Total macrophage numbers per cross-sectional area were significantly higher in testes of Inha KO mice (high activin A) than in Inhba KO (no activin A) at E13.5 and E15.5. Macrophages in Inhba KO testes were preferentially in the testis perimeter at all ages. Fluidigm revealed a significant association between activin A and mRNA levels for immune mediators that induce a pro- or anti-inflammatory microenvironment and proteins involved in migration. A reciprocal dose-dependent effect of activin A levels was identified for CX3CL1, IL-4 and IL-10 receptors, MHC class II, CCL17, CXCR7, CXCR4 and Marco transcripts. RNAseq data validated this outcome. In summary, this study demonstrates that activin A can significantly regulate testicular macrophage number and distribution pattern in the fetal testis. This may occur by modulating the synthesis of factors regulating macrophage functions.