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, Ca²⁺ independent NOS, are upregulated by E₂ in the blastocysts during implantation activation. Furthermore, p-eNOS is upregulated in implantation-induced blastocysts downstream of p-Akt.