cAMP-dependent regulation of ovulatory response genes is amplified by IGF1 due to synergistic effects on Akt phosphorylation and NF-κB transcription factors

Abstract

Granulosa cells play a crucial role as mediator of the LH-dependent ovulatory response. The intraovarian factor IGF1 is produced by ovarian somatic cells of healthy follicles during the ovulatory response. The objective of this study was to identify mechanisms by which IGF1, alone or in combination with LH, regulates the expression of genes in granulosa cells, which are crucial for ovulation. To achieve this objective, short-term, primary murine granulosa cell cultures were treated for 2-8 h with 1 mM 8-bromoadenosine 3 0 ,5 0 -cAMP to mimic the LH surge and/or 100 ng/ml IGF1. While cAMP induced significant increases in the expression of important ovulatory response genes including amphiregulin (Areg), epiregulin (Ereg), betacellulin (Btc), or interleukin 6 (Il6), IGF1 alone had no effect. However, co-treatment of cells with IGF1 and cAMP had a synergistic effect on Areg, Ereg, Btc, and Il6 mRNA abundance. Pretreatment of granulosa cells with the MEK1/2 inhibitor U0126 demonstrated that cAMP-dependent increases in Areg, Ereg, Btc, and Il6 were mediated by extracellular regulated kinase 1/2 phosphorylation. However, western blot analyses coupled with pretreatment of cells with the PI3K inhibitor LY294002 indicated that the synergistic effect of cAMP and IGF1 on transcript levels was due in part to cooperative increases in Akt phosphorylation. Western blot analyses also demonstrated that IGF1 and the combined treatment of cAMP and IGF1 decreased NF-kB p65 phosphorylation and increased NF-kB p52 levels. Together, these data indicate that IGF1 may amplify cAMP-dependent regulation of ovulatory response gene expression above an important threshold level and therefore represents a novel role for IGF1 during ovulation.

Gene expression profiling of upregulated mRNAs in granulosa cells of bovine ovulatory follicles following stimulation with hCG

“…A similar mechanism may be associated with the progesterone receptor in GC after the LH/hCG surge. Indeed, it is known that phosphorylation of AKT/p38MAPKα is increased in GC by the activation of the EGFR following binding of the EGF-like proteins, epiregulin (EREG) and amphiregulin (AREG) [ 34 ], and that EREG and AREG are induced in bovine GC after the LH/hCG surge [ 35 , 36 ]. Such EGFR-dependent activation of the AKT/p38MAPKα pathway [ 34 , 36 ] likely contributes to the phosphorylation and activation of the progesterone receptor [ 37 ].…”

“…Indeed, it is known that phosphorylation of AKT/p38MAPKα is increased in GC by the activation of the EGFR following binding of the EGF-like proteins, epiregulin (EREG) and amphiregulin (AREG) [ 34 ], and that EREG and AREG are induced in bovine GC after the LH/hCG surge [ 35 , 36 ]. Such EGFR-dependent activation of the AKT/p38MAPKα pathway [ 34 , 36 ] likely contributes to the phosphorylation and activation of the progesterone receptor [ 37 ]. Based on these observations and our findings, we hypothesize that the FKBP5 increase in bovine GC following the preovulatory LH surge may limit the EGFR-dependent AKT/p38MAPKα phosphorylation, and thus reduce progesterone receptor phosphorylation and activation, thereby attenuating GC progesterone responsiveness during ovulation and initial luteinization.…”

Altered Theca and Cumulus Oocyte Complex Gene Expression, Follicular Arrest and Reduced Fertility in Cows with Dominant Follicle Follicular Fluid Androgen Excess

“…Quantitative RT-PCR was performed to evaluate mRNA abundance in theca cells and COCs as previously reported [31] , [32] . Primers and probes for target mRNAs were designed using Primer Express 1.5 (Applied Biosystems, Foster City, CA; Sup.…”

PI3K/PTEN/Akt and TSC/mTOR signaling pathways, ovarian dysfunction, and infertility: an update

“…A synergistic effect of cAMP and IGF1 on expression of important ovulatory response genes in murine GCs has been reported. A cooperative increase in Akt phosphorylation was responsible for the effect (Mack et al 2012).…”