Regulatory Role of Nitric Oxide in Development and Hatching of Mouse Blastocysts

Abstract

To determine the regulatory role and mechanism of nitric oxide (NO) in the development and hatching of mouse blastocysts. The Kunming female mice were superovulated and then mated with mature male mice. On the day 2.5 of their pregnancy, morulae were flushed from their uterine horns with culture media. Morulae were cultured in different concentrations of N-nitro-L arginine methyl ester (L-NAME), sodium nitroprusside (SNP), or the combination of L-NAME and SNP in culture media for 48 hours. The development and hatching of blastocysts were examined on day 4 and day 5 and the total numbers of blastocyst cells and cysteinyl aspartate specific proteinase 3 (caspase 3) were observed under confocal laser scanning microscope. With the increase of the concentration of L-NAME or SNP, the hatching rate of blastocysts and the total number of blastocyst cells were significantly reduced. The addition of 10 nmol/L SNP in culture media with 5 mmol/L L-NAME significantly increased the development of blastocysts and promoted hatching of blastocysts. However, with increase of SNP concentration in culture media with 5 mmol/L L-NAME, the development and hatching rates of blastocysts were significantly decreased. L-NAME had no obvious effect on the expression of active caspase 3 in blastocyst cells. However,when being above 500 nmol/L,SNP significantly increased the expression of caspase 3 in blastocyst cells. NO plays an important role in development and hatching of mouse blastocysts. Excessively high or low NO can damage the division of blastomeres, resulting in the failure of the blastocyst development and hatching. Also, excessively high NO can lead to the apoptosis of the blastocyst cells.