Transcriptional and epigenetic status of protamine 1 and 2 genes following round spermatids injection into mouse oocytes

The use of round spermatids that are fully active at the transcriptional level to create zygotes (i.e. round spermatid injection; ROSI) raises the question regarding the downregulation of all specific genes that are transcribed from the paternal genome at fertilization. In this study, we show that protamine 1 and 2 mRNAs, which are specific to the round spermatid stage, are repressed at the two-pronuclei (6 h) and two-cell (30 h) stages postfertilization, respectively, in ROSI embryos, by distinct mechanisms. Both genes are fully methylated in round spermatids and sperm but unmethylated in oocytes. At 6 h postfertilization, the protamine 1 and 2 genes are actively demethylated, but the demethylation process happens more rapidly in ROSI than in sperm zygotes. Treatment of zygotes with trichostatin A, a histone deacetylase (HDAC) inhibitor, maintained the protamine 2 mRNAs expression up to 30 h postfertilization while the DNA methylation status of the gene is not affected. Thus, HDACs are involved in the clearance of protamine 2 mRNAs in ROSI two-cell embryos independently of the methylation status of the repressed gene. Contrastingly, HDACs are not directly involved in protamine 1 regulation since trichostatin A does not reverse the silencing of the gene in ROSI embryos at 6 h. The protamine 1 CpG island located in the coding region is actively demethylated in ROSI one-cell embryos where the gene is repressed and may contribute to the regulation of protamine 1 gene expression. The comparison with gene reprogramming occurring during nuclear transfer makes ROSI embryos an attractive model to study the mechanisms involved in gene silencing elicited by the oocyte.