Protamine1/Protamine-2 double deficient male mice. Detailed analysis of infertility using ChiP-seq on histones of sperm and scRNA-seq. on early embryos to detail the epigenetic disturbances leading to aberrant gene -reactivation.

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In this proposal, we will further investigate protamine1 and protamine2 deficient males resulting from previous grants. In our preliminary experiments, we have shown that the sperm exhibit DNA damage and transition proteins and histones remain on the DNA. However, compared to the Protamine1 or Protamine2 deficient animals, the sperm are still able to fertilize oocytes. Interestingly, the embryos arrest between the 4 and 8 cell stages. We hypothesize that the described early gene reactivation waves are altered by the altered chromatin structure (more histones and TNP's and the DNA). We will use ChIP-seq to investigate whether histone retention is specifically altered in sperm DNA. This would suggest a hierarchy in histone to protamine exchange during spermiogenesis. It is known that in wild-type animals, histones at "developmentally-important genes" are found at the sperm DNA and it is speculated that these histones are removed first after fertilization to ensure rapid reactivation of these genes. We propose to study early gene reactivation at 4 and 8 cell stages. By comparing wild type with doubleHET animals we want to analyze how this gene reactivation changes. By correlating with the ChIP-Seq dataset, we are able to correlate the impaired gene expression with aberrant histone retention. The results could provide valuable insights for patients with fertility disorders due to shifts in protamine ratio or protamine deficiency.