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.

在这项建议中，我们将进一步调查由以前的赠款导致的精蛋白1和精蛋白2缺陷男性。在我们的初步实验中，我们已经证明精子表现出DNA损伤，过渡蛋白和组蛋白保留在DNA上。然而，与精蛋白1或精蛋白2缺陷的动物相比，精子仍然能够使卵母细胞受精。有趣的是，胚胎在4到8个细胞期之间停滞。我们假设，所描述的早期基因重新激活波被改变的染色质结构(更多的组蛋白、TNP和DNA)改变。我们将使用CHIP-SEQ来研究组蛋白在精子DNA中的滞留是否发生了特定的改变。这可能表明在精子发生过程中组蛋白到鱼精蛋白的交换存在等级关系。已知在野生型动物中，在精子DNA中发现了“发育重要基因”上的组蛋白，推测这些组蛋白在受精后首先被移除，以确保这些基因迅速重新激活。我们建议研究4和8细胞阶段的早期基因重新激活。通过比较野生型和双热休克动物，我们想要分析这个基因的重新激活是如何变化的。通过与CHIP-SEQ数据集的关联，我们能够将受损的基因表达与异常的组蛋白保留相关联。这一结果可能为因鱼精蛋白比例变化或鱼精蛋白缺乏症而导致的生育障碍患者提供有价值的见解。

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.