ESA-SRB-AOTA 2019

Assessment of acrylamide generated DNA damage in sperm on fertilisation and embryo development (#700)

Nathan D Burke 1 2 3 , Elizabeth G Bromfield 1 2 4 , Jacinta H Martin 1 2 , Shaun D Roman 1 2 3
  1. The Priority Research Centre for Reproductive Biology, The University of Newcastle, Callaghan, New South Wales 2308, Australia
  2. Discipline of Biology, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, New South Wales, Australia
  3. The Priority Research Centre for Drug Development, The University of Newcastle, Callaghan, New South Wales 2308, Australia
  4. Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Spermatozoa DNA damage is intimately linked to several reproductive pathologies including paternal factor infertility. Ingested chronically, acrylamide is metabolised by CYP2E1 to a known reproductive toxicant and DNA alkylating agent, glycidamide. Glycidamide has been implicated in inducing DNA damage in sperm and acute acrylamide exposure in vivo has induced embryonic dominant lethality and chromosomal translocations. To investigate the ramifications of DNA damage on preimplantation embryogenesis, sperm were exposed to glycidamide in conditioned in vitro fertilisation (IVF) media generated by culturing immortalised epididymal cells rich in CYP2E1 with acrylamide (10mM). This novel treatment emphasised the maintenance of sperm function and no aberrations were detected in capacitation (p<0.05) or motility (~80%). Single cell gel electrophoresis revealed significant levels of DNA damage in exposed sperm compared to control sperm (p<0.001), with a 50-fold increase in the minimum DNA damage reported in all treated cells. Glycidamide treated sperm were subsequently used for IVF. The fertilisation rate of sperm with DNA damage (~92%) was unchanged compared to control cells (~93%), and over 90% of embryos from all treatments underwent cleavage, forming 2-cell embryos, by 24 hours. Preimplantation embryo development continued unimpeded in embryos fathered by sperm carrying DNA damage; culminating in over 80% of embryos from all treatments forming blastocysts at 96 hours. A subset of embryos derived from glycidamide treated sperm (~30%) exhibited delayed development at 48 and 96 hours and may reflect the impediment of embryonic genome activation in 4-cell embryos. Investigations of DNA damage in paternal pronuclei (γ-H2A.x ICC and TUNEL staining) and blastocyst stage embryos (γ-H2A.x ICC) revealed no aberrations in embryos fathered by glycidamide treated sperm. Despite extensive DNA damage, treated sperm were able to fertilise and produce developmentally competent embryos, emphasising the importance of understanding the ramifications of DNA damage on pregnancy outcomes and offspring health.