Human ovarian granulosa cells obtained from women undergoing in vitro fertilization were exposed to 15.6, 31.25, 62.5, 125, 250, 500, 1000 μM Ni2+ for 48 h. To determine the site of action of Ni2+, the granulosa cells were stimulated to produce progesterone (P) by using maximally stimulating amounts of human chorionic gonadotropin (0.1 IU/ml hCG) or dibutyryl cyclic adenosine monophosphate (1 mM db-cAMP). The luteinizing hormone (LH) analog hCG was chosen because resultant P production requires an intact membrane receptor and db-cAMP was used to test for post LH receptor defects caused by Ni2+. Progesterone content of the culture medium was determined by radioimmunoassay (RIA), and viability of the cells was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction test. Concentration-dependent depression in both hGC and db-cAMP stimulated P production was seen at 15.625 μM or higher concentration of Ni2+, which is not cytotoxic on human ovarian granulosa cells. The viability of cells was unaffected up to 31.25 μM and decreased significantly at 62.5 μM. Our results show a dose-related depression in stimulated P production of granulosa cells at a dose that does not induce significant cytotoxic action. These data indicate that the effect of Ni2+ on P production is not due to cytotoxicity, and the cellular site(s) of inhibitory action appears to be subsequent to the membrane receptor and production of cAMP.
ASJC Scopus subject areas
- Health, Toxicology and Mutagenesis