Oral Presentation ESA-SRB 2023 in conjunction with ENSA

Immortalised mouse caput epididymal epithelial cells: a model to study how parental stress is remodelling the sperm epigenome (#78)

Jessica E Mulhall 1 2 , Natalie A Trigg 3 4 , Ilana R Berntstein 1 2 , Amanda L Anderson 1 2 , Heather C Murray 5 6 , Petra Sipilä 7 , Tessa Lord 1 2 , John E Schjenken 1 2 , Brett Nixon 1 2 , David A Skerrett-Byrne 1 2
  1. Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, Newcastle, NSW, Australia
  2. Infertility and Reproduction Research Program, Hunter Medical Research Institute, Newcastle, NSW, Australia
  3. Departments of Genetics and Pediatrics , Penn Epigenetics Institute, Institute of Regenerative Medicine, and Center for Research,University of Pennsylvania Perelman School of Medicine on Reproduction and Women's Health, Philadelphia, Pennsylvania, USA
  4. Division of Neonatology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
  5. Precision Medicine Research Program, Hunter Medical Research Institute,, Newcastle, NSW, Australia
  6. School of Biomedical Sciences and Pharmacy, University of Newcastle, Newcastle, NSW, Australia
  7. Institute of Biomedicine, Research Centre for Integrative Physiology and Pharmacology, and Turku Center for Disease Modeling, University of Turku, Turku, Finland

Male infertility and paternal preconception health are key factors influencing offspring development. Both are heavily shaped during the sperm’s transit of the epididymis, a segmented tubule situated after the testes with distinct regions: initial segment, proximal caput, corpus, and distal cauda. The luminal milieu of the heavily convoluted epididymis is essential for the functional maturation of spermatozoa. This microenvironment is created by the combined secretory and resorptive activity of the epididymal epithelium, including the release of extracellular vesicles destined for maturing sperm cells, containing fertility modulating proteins and a myriad of small non-coding RNAs (sncRNAs), acting as conduits of epigenetic information. Our laboratory has previously identified the caput region as the primary site in shaping the final sncRNA profile of mature sperm, which altered following environmental stressors. To enable investigation of this intercellular communication nexus, we have applied our label-free proteomic platform to our immortalized mouse caput epididymal epithelial cell line (mECap18). We report the identification of >5,300 proteins, >75% of which were present in the proteome of in-vivo caput epididymal epithelial cells. Furthermore, key pathways associated with protein synthesis (e.g. EIF2 signalling) and cellular protection in the male reproductive tract (e.g. sirtuin signalling) were enriched in both proteomes. This comparison supports the utility of the mECap18 cell line as a tractable in-vitro model for studying caput epididymal epithelial cell function, and importantly the epididymis’ mediation of paternal stress signatures to sperm.  The glucocorticoid receptor (NR3C1), a transcription factor and known regulator of sncRNAs, is a promising orchestrater for this transmission; further study is necessary to understand this relationship. Using an acute stress exposure model on the mECap18, we have begun to investigate the effects of acute stress exposure on NR3C1 and the efficacy of therapeutic intervention to restore NR3C1 expression to pre-exposure levels.