Pronuclear epigenetic modification of protamine deficient human sperm following injection into mouse oocytes

Abstract

ABSTRACTEpigenetic abnormalities and abnormal chromatin structure in sperm may lead to male infertility. Protamine deficiency is among the disorders of chromatin structure in sperm. The study of epigenetic changes in male pronuclei is necessary since abnormal sperm is sometimes used to create embryos using assisted reproductive techniques. The present study was carried out to compare epigenetic global marks in male pronuclei derived from normal and protamine deficient sperm cells. To do so, interspecies fertilization was used to obtain the male pronucleus. Normal and protamine deficient sperm cells, which were identified by chromomycin A3 staining, were injected into mouse oocytes. Oocytes were cultured until pronuclear formation and were then labeled with different antibodies (anti 5-methylcytosine, anti 5-hydroxymethylcytosine, and anti acetyl H4K12). Based on the fluorescence intensity, the level of each of these epigenetic factors was determined and they revealed a significant relationship between the level of sperm protamine deficiency and sperm epigenetic factors. Protamine deficiency was found to be associated with an increased methylation (p=0) and decreased hydroxymethylation rate (p=0.015) of the male pronucleus chromatin. However, no association was found between protamine deficiency and the level of H4K12 acetylation (p=0.548). Also, the efficiency of fertilization in protamine deficient sperm cells was less than normal. These results suggest that protamine deficient sperm cells lead to the formation of epigenetically altered pronuclei.