Nitric oxide regulates blastocyst hatching in mice.

OBJECTIVE: This study is to determine the regulatory role of nitric oxide in mouse blastocyst hatching. METHODS: Kunming female mice were superovulated and then mated with mature male mice. On day 2.5 of their pregnancy, the pregnant mice were killed and morulae were flushed from their uterine horns with culture media. Morulae were cultured in media with different concentrations of N-nitro-L arginine methyl ester (L-NAME), sodium nitroprusside (SNP), 8-Br-3'-5'-cyclic guanosine monophosphate (8-Br-cGMP) or the combination of L-NAME with SNP or 8-Br-cGMP for 48 h. The hatched blastocysts were examined on day 5 and the expressions of epithelial nitric oxide synthase (eNOS) and active cysteinyl aspartate specific proteinase 3 (caspase 3) were observed under confocal laser scanning microscope. RESULTS: L-NAME significantly reduced the expression of eNOS in blastocyst cells. With the increase of the concentrations of L-NAME, SNP or 8-Br-cGMP, blastocyst hatching rate was significantly lowered. In addition, 5 mM L-NAME, 2 μM SNP and 2 μM 8-Br-cGMP completely inhibited blastocyst hatching. Low concentrations of SNP or 8-Br-cGMP in culture media containing 5 mM L-NAME significantly reversed the inhibition of blastocyst hatching and promoted hatching development. Moreover, 5 mM L-NAME and 2 μM 8-Br-cGMP had no significant influence on the expression of active caspase 3 in blastocyst cells. SNP (> 500 nM) significantly increased the expression of active caspase 3 in blastocyst cells. CONCLUSIONS: NO/cGMP pathway plays an important role in mouse blastocyst hatching. Excessive or depleted NO can interrupt blastocyst hatching. Excessive NO leads to apoptosis of blastocyst cells.