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 I 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 prolamine I 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 prolamine 2 mRNAs in ROSI two-cell embryos independently of the methylation status of the repressed gene. Contrastingly, HDACs are not directly involved in protamine I regulation since trichostatin A does not reverse the silencing of the gene in ROSI embryos at 6 h. The protamine I 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 I 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. (c) 2007 Elsevier Inc. All rights reserved.