Upregulation of iNOS and phosphorylated eNOS in the implantation‐induced blastocysts of mice

PURPOSE: This study aimed to examine expressions of iNOS and phosphorylated eNOS (p‐eNOS) in implantation‐induced blastocysts. We also examined the upstream of p‐eNOS. METHODS: To address the protein expressions in implantation‐induced blastocysts, we performed immunohistochemical analysis using a delayed implantation mouse model. Immunostaining for iNOS, p‐eNOS, and p‐Akt was done. To address the relationship between p‐eNOS and p‐Akt, activated blastocysts were treated with an Akt inhibitor, MK‐2206. RESULTS: iNOS expression was at low levels in dormant blastocysts, whereas the expression was significantly increased in the activated blastocysts. Double staining of p‐eNOS and p‐Akt in individual blastocysts showed colocalization of p‐eNOS and p‐Akt of the trophectoderm. p‐eNOS and p‐Akt expressions were at low levels in dormant blastocysts, whereas both of them were significantly increased in the activated blastocysts. Both dormant and activated blastocysts showed significant positive correlations between p‐eNOS and p‐Akt. MK‐2206 treatment for activated blastocysts showed that blastocysts with lower p‐Akt had significantly lower p‐eNOS levels. CONCLUSIONS: iNOS and p‐eNOS, Ca(2+) independent NOS, are upregulated by E(2) in the blastocysts during implantation activation. Furthermore, p‐eNOS is upregulated in implantation‐induced blastocysts downstream of p‐Akt.