Nitric oxide regulates steroid synthesis by bovine antral granulosa cells in a chemically defined medium

• Published in: Animal reproduction science 2009-02, Vol.110 (3), p.222-236
• Main Authors: Faes, M.R., Caldas-Bussiere, M.C., Viana, K.S., Dias, B.L., Costa, F.R., Escocard, R.M.
• Format: Article
• Language: English
• Subjects: 17β-Estradiol; Animals; apoptosis; biochemical pathways; Cattle; Cattle - metabolism; cell culture; Cell Cycle - drug effects; Cell Cycle - physiology; Cell Survival - drug effects; cGMP; cows; Cyclic GMP - antagonists & inhibitors; Cyclic GMP - metabolism; cyclic guanosine monophosphate; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; Estradiol - biosynthesis; estrogens; Female; follicular development; Granulosa cells; Granulosa Cells - drug effects; Granulosa Cells - metabolism; guanosine monophosphate; in vitro studies; mitochondria; Nitrates - analysis; Nitric oxide; Nitric Oxide - metabolism; Nitric Oxide Donors - pharmacology; Nitroprusside - pharmacology; Oxadiazoles - pharmacology; physiological regulation; Progesterone; Progesterone - biosynthesis; Quinoxalines - pharmacology; signal transduction; sodium nitroprusside; steroidogenesis
• ISSN: 0378-4320; 1873-2232
• DOI: 10.1016/j.anireprosci.2008.01.018

Nitric oxide (NO) in bovine ovary has been characterized as one of the controllers of granulosa cells’ (GC) steroidogenesis and apoptosis. One of the pathways used by NO to have these effects is cGMP. The objectives of the present study were to verify the effect of sodium nitroprusside (SNP), a NO donor, on steroidogenesis, cell viability (mitochondrial activity) and GC cell cycle distribution and if this effect occurs by the NO-cGMP signaling pathway with the addition of SNP with or without 1H-[1,2,3] oxadiaziolo[4,3a]quinoxaline-1-one (ODQ), a selective soluble guanylate cyclase inhibitor. The antral GC from 3 to 5 mm diameter cattle follicles was cultured without treatment (control), with ODQ (10 −4 M) and 10 −5, 10 −3 and 10 −1 M SNP with or without ODQ for 24 h. Nitrate/nitrite (NO 3 −/N0 2 −) concentrations were evaluated by Griess method, progesterone (P 4) and 17β-estradiol (E 2) concentrations by chemiluminescence, viability and cell cycle stage by MTT method (3-[4,5-dimethylthiazol-2yl]-2,3 dipheniltetrazolium bromide) and flow cytometry, respectively. Nitrate/nitrite concentration in culture medium increased ( P < 0.05) in a dose-dependent manner according to SNP concentration added to the culture medium. The GC cultured without treatment, with ODQ and with 10 −5 M SNP in the presence or absence of ODQ developed into cell aggregates and did not vary in cell viability ( P > 0.05), while GC cultured with 10 −3 and 10 −1 M SNP with or without ODQ presented disorganized GC aggregates or did not develop into cell aggregates and also had substantially decreased cell viability (mitochondrial activity inhibition) and steroids synthesis ( P < 0.05), and effects were not reversed with us of ODQ. Most GC cultured without treatment (control) or with ODQ, 10 −5 and 10 −3 M SNP with or without ODQ were in the G0/G1 (80–75%) stage and in a lesser proportion (20–25%) in the S + G2/M stage of the cell cycle, while the 10 −1 M SNP treatment resulted in GC in G1 phase arrest. The treatment with 10 −5 M SNP increased ( P < 0.05) E 2 synthesis and inhibited ( P < 0.05) progesterone synthesis. The addition of ODQ reversed ( P < 0.05) the stimulatory effect of 10 −5 M SNP treatment on E 2, but not on P 4 synthesis ( P > 0.05). These results demonstrated that E 2 synthesis by antral GC from small follicles is modulated by lesser NO concentrations via the cGMP pathway, but not P 4 while steroids inhibition cGMP pathway independent, mitochondrial damage and the interfere