Expression of endothelial nitric oxide synthase gene in cultured porcine granulosa cells after FSH stimulation

The present study was designed to investigate nitric oxide (NO) synthesis a nd the expression of endothelial NO synthase (eNOS) gene in cultured porcin e granulosa cells. Granulosa cells prepared from small follicles (1-4 mm di ameter) mere cultured in plastic dishes coated with fibronectin in chemical ly defined medium, and matured after 48 h of stimulation with FSH. The conc entrations of nitrite and nitrate remained relatively constant until 42 h o f stimulation, after which they increased significantly up to twofold at 48 h. NO synthesis was accompanied by. an increase in cGMP. Gene expression f or eNOS was studied by RT-PCR, and a PCR product of the expected size ampli fied. eNOS mRNA was expressed in the presence of FSH, but not in the absenc e of FSH. Although eNOS mRNA was not expressed in the initial period, it wa s expressed after 12 h of stimulation with FSH, and remained at a relativel y constant level until 48 h. Expression of eNOS mRNA preceded expression of LH receptor mRNA, which showed a maximal level at 24 h of stimulation. The se observations suggest that eNOS expression is not related to a rapid synt hesis of NO in developing granulosa cells, and that the activation of NO sy nthesis is rigidly regulated in the initial period of development.