Copper and Selenium stimulates CYP19A1 expression in caprine ovarian granulosa cells: possible involvement of AKT and WNT signalling pathways

The role of copper and selenium on activation of estradiol synthesis pathways viz. PKA/AKT/WNT is not clearly elucidated. On this background we attempt to elcuiated the role of copper and selenium on mRNA expression of genes associated with estradiol synthesis in caprine ovarian granulose cell models. Ovarian granulosa cells from medium (3-5 mm) sized follicles were aspirated and distributed separately to different groups. Group I: control, Group II: cupric chloride (Cu: 0.5 mM), Group III: sodium selenite (Se: 100 ng/ml), Group IV: Cu + Se. The cells (10(5)/well) were cultured in 96 well plate in the base culture medium of MEM alpha comprising of nonessential amino acids (1.1 mM), FSH (10 ng/mL), transferrin (5 mu g/mL), IGF-I (2 ng/mL), androstenedione (10(-6) M), penicillin (100 IU/mL), streptomycin (0.1 mg/mL) and fungizone (0.625 mu l/mL) and insulin (1 ng/mL). The cells were incubated in a carbondioxide incubator (38 degrees C, 5% CO2, 95% RH). The medium was changed on alternate days and cells were harvested on day 6. Day 6 media was used for estimation of estradiol. The RNA isolated form harvested cells was used for qPCR assay. There was no significant (p > 0.05) difference in estradiol concentration between groups. The mRNA expression of AKT1, CYP19A1, WNT2 & 4, FZD6 and APC2 were significantly (p < 0.05) higher in Cu and Cu + Se groups compared to control. Whereas, the mRNA transcript of DVL1 and CSNK1 was significantly (p < 0.05) higher in Cu + Se group compared to control. Incontrast, no significant difference in mRNA expression of PRKAR1A and CTNNB1 was noticed. Our study support a key role of copper and selenium in activation of AKT and WNT signalling pathway that further lead to increase in the mRNA expression of CYP19A1.