Nitric oxide regulation of preimplantation embryo development

Abstract

Objective: To determine if multiple nitric oxide synthase (NOS) isoforms are expressed by preimplantation embryos and to evaluate the effect of modulating NO production on embryo development and the induction of apoptosis. Design: Research study. Materials/Methods: Mouse embryos and blastocysts were obtained from CD-1 female mice mated with males of the same strain (day 1 = vaginal plug). Day 1 embryos were flushed from excised oviducts and cultured in Whitten’s culture medium for 24–96 hours. RT-PCR was performed to determine the presence of NOS mRNA in 2-cell, 4-cell, morula, and blastocyst stage embryos. A LightcyclerTM, a combination microvolume fluorimeter and rapid temperature cycler (Roche, Indianapolis, IN USA) was used for this analysis. Melting curves were plotted to determine PCR product identity. Product identity was confirmed by ethidium bromide stained 2% agarose gel electrophoresis. To determine if altered NO production initiated apoptosis, comet assay was performed on day-1 embryos cultured for 72 h in either Whittens media or Whittens with one of the following: 500 uM L-NA, 200 uM 8-Br-cGMP, or 250 uM NO donor, S-nitroso-N-acetylpenicillamine (SNAP). Results: The presence of multiple NOS isoforms was detected in preimplantation embryos. However, they are not expressed at the same the level throughout development. Embryos cultured in Whittens, 500 uM L-NA, and 200 uM 8-Br-cGMP showed little or no DNA damage whereas embryos cultured with 250 uM SNAP showed extensive DNA damage. Conclusions: Nitric oxide (NO) production plays an important role in regulating pre-implantation embryo development. It has been previously shown that inhibition of NO production by NG-nitro-L-arginine (L-NA) inhibits the development of preimplantation embryos from the 2-cell to the blastocyst stage (Gourge et al., Biol Reprod 1998 4:875–879). However, excess NO also halts embryo development at the 2-cell stage, possibly through the production of free radicals, as culture with 8-Br-cGMP did not induce apoptosis. This study demonstrates the presence of multiple NOS isoforms in preimplantation embryos, providing further evidence for the importance of nitric oxide production in embryo development. Furthermore, we have established that increased NO production induces apoptosis in embryos while lack of NO inhibits development but does not induce cell death. Together, these data directly demonstrate the importance of NO in preimplantation embryos and furthers our understanding of altered development as a result of both NO inhibition and excess NO. Supported by: NIH. ART Institute of NY/NJ. ART Institute of Washington.