Influence of Nitric Oxide on Steroid Synthesis, Growth and Apoptosis of Buffalo (Bubalus bubalis) Granulosa Cells In vitro

  • Dubey, Pawan K. (Reproductive Physiology and ETT Laboratory, Physiology and Climatology Division, Indian Veterinary Research Institute) ;
  • Tripathi, Vrajesh (Department of Animal Science, M.J.P. Rohilkhand University) ;
  • Singh, Ram Pratap (Division of Physiology and Reproduction, Central Avian Research Institute) ;
  • Sastry, K.V.H. (Division of Physiology and Reproduction, Central Avian Research Institute) ;
  • Sharma, G.Taru (Reproductive Physiology and ETT Laboratory, Physiology and Climatology Division, Indian Veterinary Research Institute)
  • Received : 2010.08.03
  • Accepted : 2010.12.28
  • Published : 2011.09.01


Objective of this study was to examine the effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor on steroid synthesis, growth and apoptosis of buffalo granulosa cells (GCs) in vitro. Follicular fluid of antral follicles (3-5 mm diameter) was aspirated and GCs were cultured in 0 (control), $10^{-3}$, $10^{-5}$, $10^{-7}$, $10^{-9}\;M$ of SNP for 48 h. To evaluate whether this effect was reversible, GCs were cultured in presence of $10^{-5}\;M$ SNP+1.0 mM $N^{\omega}$-nitro-L-arginine methyl ester (L-NAME) a NO synthase (NOS) inhibitor or hemoglobin (Hb, $1.0{\mu}g$) as NO scavenger. Nitrate/nitrite concentration was evaluated by Griess method, progesterone and estradiol concentrations by RIA and apoptosis by TUNEL assay. SNP ($10^{-3}$, $10^{-5}$, $10^{-7}\;M$) significantly (p<0.05) inhibited estradiol and progesterone synthesis, growth, disorganized GCs aggregates and induced apoptosis in a dose dependent manner. However, $10^{-9}\;M$ SNP induced the progesterone synthesis and stimulated GCs to develop into a uniform monolayer. Combination of SNP $10^{-5}$ M+L-NAME strengthened the inhibitory effect while, SNP+Hb together reversed these inhibitory effects. In conclusion, SNP at greater concentrations ($10^{-3}$, $10^{-5}$ and $10^{-7}\;M$) has a cytotoxic effect and it may lead to cell death whereas, at a lower concentration ($10^{-9}\;M$) induced progesterone synthesis and growth of GCs. These findings have important implications that NOS derived NO are involved at physiological level during growth and development of buffalo GCs which regulates the steroidogenesis, growth and apoptosis.


  1. Basini, G., M. Baratta, N. Ponderato, S. Bussolati and C. Tamanini. 1998. Is nitric oxide an autocrine modulator of bovine granulosa cell function? Reprod. Fertil. Dev. 10:471-478.
  2. Chaube, S. K., K. D. Pawan, K. M. Surabhi and T. G. Shrivastav. 2007. Verapamil reversibly inhibits spontaneous parthenogenetic activation in aged rat eggs cultured in vitro. Cloning Stem Cells 9:615-624.
  3. Chun, S. Y., K. M. Eisenhauer, M. Kubo and A. J. W. Hsueh. 1995. Interleukin-1$\beta$ suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production. Endocrinology 136:3120-3127.
  4. Ellman, C., J. A. Corbett and T. P. Misko. 1993. Nitric oxide mediates interleukin-1 induced cellular cytotoxicity in the rat ovary. J. Clin. Invest. 92:3053-3056.
  5. Faes, M. R., M. C. Caldas-Bussiere, K. S. Viana, B. L. Dias, F. R. Costa and R. M. Escocard. 2009. Nitric oxide regulates steroid synthesis by bovine antral granulosa cells in a chemically defined medium. Anim. Reprod. Sci. 110:222-236.
  6. Goud, P. T., A. P. Goud, P. Michael, B. G. Diamond, M. Husam and A. Soud. 2008. Nitric oxide extends the oocyte temporal window for optimal fertilization. Free Radic. Biol. Med. 45:453-459.
  7. Hsueh, A. J. W., H. Billig and A. Tsafriri. 1994. Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocrinological Review 15:707-723.
  8. Ignarro, L. J., R. G. Harbison, K. S. Wood and P. J. Kadowitz. 1986. Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid. J. Pharmacol. Exp. Ther. 237:893-900.
  9. Jablonka-Shariff, A. and L. M. Olson. 2000. Nitric oxide is essential for optimal meiotic maturation of murine cumulusoocyte complexes in vitro. Mol. Reprod. Dev. 55:412-442.<412::AID-MRD9>3.0.CO;2-W
  10. Jablonka-Shariff, A. and L. M. Olson. 1997. Hormonal regulation of nitric oxide synthases and their cell-specific expression during follicular development in the rat ovary. Endocrinology 138:460-468.
  11. Jablonka-Shariff, A., R. Basuray and L. M. Olson. 1999. Inhibitors of nitric oxide synthase influence oocyte maturation in rats. J. Soc. Gynecol. Investig. 6:95-101.
  12. Jurisicova, A., K. E. Latham, R. F. Casper and S. L. Varmuza. 1998. Expression and regulation of genes associated with cell death during murine preimplantation embryo development. Mol. Reprod. Dev. 51:243-253.<243::AID-MRD3>3.0.CO;2-P
  13. Kaneto, H., J. Fujn and H. G. Seo. 1995. Apoptoac cell death tnggered by nitnc oxide in pancreatic P-cells. Diabetes 44: 733-738.
  14. Masuda, M. T., S. Kubota and T. Kamada. 1997. Nitric oxide inhibits steroidogenesis in cultured porcine granulosa cells. Mol. Hum. Reprod. 3:285-292.
  15. Matta, S. G., M. C. Caldas-Bussiere, K. S. Viana, M. R. Faes, C. S. Paes de Carvalho, B. L. Dias and C. R. Quirino. 2009. Effect of inhibition of synthesis of inducible nitric oxide synthasederived nitric oxide by aminoguanidine on the in vitro maturation of oocyte-cumulus complexes of cattle. Anim. Reprod. Sci. 111:189-201.
  16. Moncada, S., R. M. J. Palmer and E. A. Higgs. 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacological Review 43:109-142.
  17. Olson, L. M., C. M. Jones-Burton and A. Jablonka-Shariff. 1996. Nitric oxide decreases estradiol synthesis of rat luteinized ovarian cells: Possible role for nitric oxide in functional luteal regression. Endocrinology 137:3531-3539.
  18. Orsi, N. M. 2006. Embryotoxicity of the nitric oxide donor sodium nitroprusside in preimplantation bovine embryons in vitro. Anim. Reprod. Sci. 91:225-236.
  19. Pires, P. R., N. P. Santos, P. R. Adona, M. M. Natori, K. R. Schwarz, T. H. de Bem and C. L. Leal. 2009. Endothelial and inducible nitric oxide synthases in oocytes of cattle. Anim. Reprod. Sci. 116:233-243.
  20. Richter, C. 1999. Pro-oxidants and mitochondrial Ca+2: their relationship to apoptosis and oncogenesis. FEBS Lett. 325: 104-107.
  21. Rosselli, M., P. J. Keller and R. K. Dubey. 1998. Role of nitric oxide in the biology, physiology and pathophysiology of reproduction. Hum. Reprod. Update 4:3-24.
  22. Salvemini, D. 1997. Regulation of cyclooxygenase enzymes by nitric oxide. Cell. Mol. Life Sci. 53:576-582.
  23. Sarti, P., E. Lendaro, R. Ippoliti, A. Bellelli, P. A. Benedetti and M. Brunori. 1999. Modulation of mitochondrial respiration by NO: investigation by single cell fluorescence microscopy. FASEB J. 13:191-197.
  24. Sastry, K. V. H., R. P. Moudgal, J. Mohan, J. S. Tyagi and G. S. Rao. 2002. Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy. Anal. Biochem. 306:79-82.
  25. Schwarz, K. R. L., P. R. L. Pires, T. H. C. de Bem, P. R. Adona and C. L. V. Leal. 2010. Consequences of nitric oxide synthase inhibition during bovine oocyte maturation on meiosis and embryo development. Reprod. Domest. Anim. 45:75-80.
  26. Schwarz, K. R. L., R. L. Pedro, P. Pires, R. Adona, H. Tiago, B. D. Camara, L. Claudia and V. Leal. 2008. Influence of nitric oxide during maturation on bovine oocyte meiosis and embryo development in vitro. Reprod. Fertil. Dev. 20:529-536.
  27. Snyder, G. D., R. W. Holmes, J. N. Bates and B. J. Van Voorhis. 1996. Nitric oxide inhibits aromatase activity: mechanisms of action. J. Steroid Biochem. Mol. Biol. 58:63-69.
  28. Tanner, F. C., P. Meier, H. Greutert, C. Champion, E. G. Nabel and T. F. Luscher. 2000. Nitric oxide modulates expression of cell cycle regulatory proteins: a cytostatic strategy for inhibition of human vascular smooth muscle cell proliferation. Circulation 101:1982-1989.
  29. Tao, Y., Z. Fu, M. J. Zhang, G. L. Xia, J. Yang and H. R. Xie. 2004. Immunohistochemical localization of inducible and endothelial nitric oxide synthase in porcine ovaries and effect of NO on antrum formation and oocyte maturation. Mol. Cell. Endocrinol. 222:93-103.
  30. Tamanini, C., G. Basin, F. Grasselli and M. Tirelli. 2003. Nitric oxide and the ovary. J. Anim. Sci. 81:E1-E7.
  31. Tilly, J. L., K. I. Tilly, M. L. Kenton and A. L. Johnson. 1995. Expression of members of the bcl-2 gene family m the immature rat ovary equine chononic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-x long messenger nbonucleic acid levels. Endocrinology 136:232-241.
  32. Van Voorhis, B. J., M. S. Dunn, G. D. Snyder and C. P. Weiner. 1994. Nitric oxide: an autocrine regulator of human granulosaluteal cell steroidogenesis. Endocrinology 135:1779-1806.
  33. Wink, D. A., Y. Osawa, J. F. Darbyshire, C. R. Jones, S. C. Eshenaur and R. W. Nims. 1993. Inhibition of cytochromes progesterone 50 by nitric oxide and a nitric oxide-releasing agent. Arch. Biochem. Biophys. 300:115-123.
  34. Yang, D., U. Lang and S. G. Greenberg. 1996. Elevation of nitrate levels in pregnant ewes and their fetuses. Am. J. Obstet. Gynecol. 174:573-577.