[Introduction]The parental genomes in zygotes undergo 5-methylcytosine (5mC) oxidation by a dioxygenase TET3, which converts 5mC to 5-hydroxycytosine (5hmC). However, its interacting proteins have not been identified yet. We have reported that maternal gonad specific expression gene (1700011E24RIK or GSE) is essential for 5mC oxidation in mouse zygotes. To understand how GSE protein is involved in 5mC oxidation, we sought to identify the maternal factors interacting with GSE in mouse zygotes. [Methods]To investigate the interaction of GSE-TET3 and identify its interacting protein, we performed yeast two-hybrid system using cDNA library of MII oocytes and ovaries and co-immunoprecipitation using PN3 zygotes. We performed immunostaining of 5mC and 5hmC and methylated DNA immunoprecipitation (MeDIP) and hydroxymethylated DNA immunoprecipitation (hMeDIP) analysis in Line1 retrotransposon sequence using knockdown (KD) by injecting the siRNA and knockout (KO) zygotes at PN3 and 5 stages. We treated TBBD, a specific inhibitor of methylation at H3R17, in growing oocytes and collected zygotes by performing in vitro maturation and intra cytoplasmic sperm injection (ICSI).[Results and Discusion]We showed that GSE interacted with TET3 and maternal methyltransferase like 23 (METTL23), a novel arginine methyltransferase that catalyzed asymmetric dimethylated histone H3 arginine 17 (H3R17me2a). Knockout of GSE or KD of METTL23 resulted in significant reduction of 5mC oxidation, as demonstrated by an increased 5mC level and a decreased 5hmC level in the male pronucleus. This was most likely due to deficient pronuclear localization of TET3. The impaired 5mC oxidation was confirmed by MeDIP and hMeDIP in Line1 in zygotes derived from maternal GSE- or METTL23-KD. Treatment with TBBD reduced the H3R17me2a level in the both pronuclei, and caused higher 5mC and lower 5hmC levels in the male pronucleus. Thus, TET3-dependent 5mC oxidation in zygotes is regulated by specific histone modification H3R17me2a.