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Asian-Australas J Anim Sci > Volume 24(9); 2011 > Article
Animal Reproduction and Physiology
Asian-Australasian Journal of Animal Sciences 2011;24(9): 1204-1210.
DOI: https://doi.org/10.5713/ajas.2011.10290    Published online August 24, 2011.
Influence of Nitric Oxide on Steroid Synthesis, Growth and Apoptosis of Buffalo (Bubalus bubalis) Granulosa Cells In vitro
Pawan K. Dubey, Vrajesh Tripathi, Ram Pratap Singh, K. V. H. Sastry, G. Taru Sharma
Abstract
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-nitro-L-arginine methyl ester (L-NAME) a NO synthase (NOS) inhibitor or hemoglobin (Hb, 1.0 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.
Keywords: Apoptosis; Buffalo; Granulosa Cell; Nitric Oxide; Steroidogenesis


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