Participation of the mouse implanting trophoblast in nitric oxide production during pregnancy

While considerable progress has been made in elucidating nitric oxide (NO) regulatory mechanisms in the later stages of gestation, much less is known about its synthesis and role during embryo implantation. Thus, to evaluate the participation of the trophoblast in the production of NO during this ph ase, this study focused on NADPH-diaphorase activity and the distribution o f NO synthase isoforms (NOS) using immunohistochemistry in pre- and postimp lantation mouse embryos in situ and in vitro, as well as on NO production i tself, measured as total nitrite, in trophoblast culture supernatants (Grie ss reaction). No NADPH-diaphorase activity was found in preimplanting embry os except alter culturing for at least 48 h, when a few trophoblastic giant cells were positive. Conversely, postimplantation trophoblast cells either lodged into the implantation chamber (in situ) or after culturing (in vitr o) showed intense NADPH-diaphorase activity. Also in the postimplantation t rophoblast, the endothelial and inducible NOS (eNOS and iNOS) isoforms were immunodetected, under both in situ and in vitro conditions, although in di fferent patterns. Extracts of ectoplacental cone also revealed bands of 135 and 130 kDa on SDS-PACE that reacted with anti-eNOS and anti-iNOS, respect ively, on Western blot. Analysis of the culture supernatant demonstrated th at the nitrite concentration was 1) proportional to the number of cultured tropho-blast cells, 2) almost completely abolished in the presence of N-ome ga-nitro-L-arginine methyl ester, and 3) increased 2-fold in cultures stimu lated with gamma-interferon, These results strongly suggest the production of NO from constitutive and inducible isoforms of NOS by the implanting mou se trophoblast, They also emphasize the possibility of the participation of these cells in vasodilatation and angiogenesis, and in cytotoxic mechanism s involved in the intense phagocytosis of injured maternal cells, which occ ur during the implantation process.