Abstract

ObjectivesDuring ovulation, it has been shown that LH stimulus induces the expression of numerous genes via PKA, p38 MAPK, PI3K and ERK1/2 in cumulus cells and granulosa cells. Our recent study showed that EGF-like factor and its protease (TACE/ADAM17) are required for the activation of EGF receptor (EGFR), cumulus expansion and oocyte maturation of porcine cumulus-oocyte complexes (COCs). In the present study, we investigated which signaling pathways are involved in the gene expression of EGF-like factor and in Tace/Adam17 expression in cumulus cells of porcine COC during in vitro maturation.MethodsAreg, Ereg, Tace/Adam17, Has2, Tnfaip6 and Ptgs2 mRNA expressions were detected in cumulus cells of porcine COCs by RT-PCR. Protein level of ERK1/2 phosphorylation in cultured cumulus cells was analyzed by westernblotting. COCs were visualized using a phase-contrast microscope.ResultsWhen COCs were cultured with FSH and LH up to 2.5 h, Areg, Ereg and Tace/Adam17 mRNA were expressed in cumulus cells of COCs. Areg, Ereg and Tace/Adam17 gene expressions were not suppressed by PI3K inhibitor (LY294002), whereas PKA inhibitor (H89), p38 MAPK inhibitor (SB203580) and MEK inhibitor (U0126) significantly suppressed these gene expressions. Phosphorylation of ERK1/2, and the gene expression of Has2, Tnfaip6 and Ptgs2 were also suppressed by H89, SB203580 and U0126, however, these negative effects were overcome by the addition of EGF to the medium, but not in the U0126 treatment group.ConclusionThe results showed that PKA, p38 MAPK and ERK1/2 positively controlled the expression of EGF-like factor and TACE/ADMA17, the latter of which impacts the cumulus expansion and oocyte maturation of porcine COCs via the EGFR-ERK1/2 pathway in cumulus cells.

Highlights

  • In mammals, luteinizing hormone (LH) stimulation induces morphological and physiological changes in granulosa cells and cumulus cells, causing them to progress to the ovulation process [1]

  • Effect of each specific inhibitor of protein kinase A (PKA), p38 p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and MEK on the gonadotropin-induced Areg, Ereg and Tace/Adam17 mRNA expression during in vitro maturation of porcine cumulus-oocyte complex (COC) COCs were cultured with follicle stimulating hormone (FSH), LH and/or PKA inhibitor (H89), p38 MAPK inhibitor (SB203580), MEK inhibitor (U0126) or PI3K inhibitor (LY294002) for 2.5 h

  • The results showed that high levels of Areg, Ereg and Tace/ Adam17 mRNA were observed when COCs were cultured with FSH and LH, and that the levels were not affected by LY294002 (Figure 1A, B, C)

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Summary

Introduction

In mammals, luteinizing hormone (LH) stimulation induces morphological and physiological changes in granulosa cells and cumulus cells, causing them to progress to the ovulation process [1]. During this period, cumulus cells expressed cumulus expansion-related genes, Hyaluronan synthase 2 (Has2) [2,3], Tumor necrosis factor αinduced protein 6 (Tnfaip6) [4,5], and Pentraxin 3 (Ptx3) [6,7], which is necessary for the synthesis and stability of hyaluronan-rich extracellular matrix. In vivo during the ovulation process, LH induces EGF-like factor expression in granulosa cells and the release of the EGF domain by TACE/ADAM17 acts on cumulus cells, which induce cumulus expansion

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