Prostaglandin endoperoxide synthase 2 (PTGS2), tumor necrosis factor-alpha-induced protein-6 (TNFAIP6), pentraxin-3 (PTX3), epidermal growth factor like factors: amphiregulin (AREG) and epiregulin (EREG) are essential for successful ovulation. We compared here the induction of these ovulatory genes in bovine granulosa cells (GCs) in vivo (after LH surge) and in vitro (forskolin treatment). These genes were markedly stimulated in GCs isolated from cows 21hr after LH- surge. In isolated GCs, forskolin induced a distinct temporal profile for each gene. Generally, there was a good agreement between the in vivo and in vitro inductions of these genes except for PTX3. Lack of PTX3 induction in isolated GCs culture suggests that other follicular compartments may mediate its induction by LH. Next, to study the role of PTGS2 and prostaglandins in the cascade of ovulatory genes, PTGS2 was silenced with small interfering RNA (siRNA). PTGS2 siRNA caused a marked and specific knockdown of PTGS2 mRNA and PGE2 production (70% compared to scrambled siRNA) in bovine GCs. Importantly, PTGS2 silencing also reduced AREG, EREG and TNFAIP6 mRNA but not PTX3. Exogenous PGE2 increased AREG, EREG and TNFAIP6 mRNAs, further confirming that these genes are prostanoid-dependent. A successful and specific knockdown of PTGS2 was also achieved in endometrial cells (EndoCs) expressing PTGS2. Then, cholesterol-conjugated PTGS2 (chol-PTGS2) siRNA that facilitates cells' entry was examined. In EndoCs, but not in GCs, Chol-PTGS2 siRNA succeeded to reduce PTGS2 and PGE2 levels even without transfection reagent. PTGS2 knockdown is a promising tool to critically examine the functions of PTGS2 in the reproductive tract.