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

Abstract

The present study was designed to investigate nitric oxide (NO) synthesis and the expression of endothelial NO synthase (eNOS) gene in cultured porcine granulosa cells. Granulosa cells prepared from small follicles (1-4 mm diameter) were cultured in plastic dishes coated with fibronectin in chemically defined medium, and matured after 48 h of stimulation with FSH. The concentrations of nitrite and nitrate remained relatively constant until 42 h of stimulation, after which they increased significantly up to twofold at 48 h. NO synthesis was accompanied by an increase in cGMP. Gene expression for eNOS was studied by RT-PCR, and a PCR product of the expected size amplified. eNOS mRNA was expressed in the presence of FSH, but not in the absence of FSH. Although eNOS mRNA was not expressed in the initial period, it was expressed after 12 h of stimulation with FSH, and remained at a relatively 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. These observations suggest that eNOS expression is not related to a rapid synthesis of NO in developing granulosa cells, and that the activation of NO synthesis is rigidly regulated in the initial period of development.