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Activation of PKA, p38 MAPK and ERK1/2 by gonadotropins in cumulus cells is critical for induction of EGF-like factor and TACE/ADAM17 gene expression during in vitro maturation of porcine COCs.

Yamashita Y, Hishinuma M, Shimada M - J Ovarian Res (2009)

Bottom Line: 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.The 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Veterinary Medicine, Faculty of Agriculture, Tottori University, 4-101 Koyamachou-minami, Tottori, 680-8553, Japan.

ABSTRACT

Objectives: During 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.

Methods: Areg, 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.

Results: When 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.

Conclusion: The 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.

No MeSH data available.


Effect of H89, SB203580 or U0126 on expression of Has2(A), Tnfaip6(B) or Ptgs2(C) mRNA. For reference, the 0 h COC value was set as 1 and the data presented as the fold strength. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 10 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 10 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH, SB230580 and EGF for 10 h. The respective value of among Has2, Tnfaip6 and Ptgs2 mRNA were normalized according to those of β-actin mRNA to evaluate arbitrary units of the relative abundance of the targets. FSH(-): COCs were cultured without FSH and LH for 10 h; FSH(+): COCs were cultured with FSH and LH for 10 h; EGF(-): COCs were cultured without EGF for 10 h; EGF(+): COCs were cultured with EGF for 10 h; +H89: COCs were cultured with H89 for 10 h; +SB: COCs were cultured with SB203580 for 10 h; +U0126: COCs were cultured with U0126 for 10 h
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Figure 3: Effect of H89, SB203580 or U0126 on expression of Has2(A), Tnfaip6(B) or Ptgs2(C) mRNA. For reference, the 0 h COC value was set as 1 and the data presented as the fold strength. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 10 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 10 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH, SB230580 and EGF for 10 h. The respective value of among Has2, Tnfaip6 and Ptgs2 mRNA were normalized according to those of β-actin mRNA to evaluate arbitrary units of the relative abundance of the targets. FSH(-): COCs were cultured without FSH and LH for 10 h; FSH(+): COCs were cultured with FSH and LH for 10 h; EGF(-): COCs were cultured without EGF for 10 h; EGF(+): COCs were cultured with EGF for 10 h; +H89: COCs were cultured with H89 for 10 h; +SB: COCs were cultured with SB203580 for 10 h; +U0126: COCs were cultured with U0126 for 10 h

Mentions: High levels of Has2, Tnfaip6 and Ptgs2 expression were observed in cumulus cells of COCs cultured for 10 h with FSH and LH as compared with those cultured without FSH and LH. The addition of H89, SB20580 or U0126 significantly suppressed FSH- and LH-induced Has2, Tnfaip6 and Ptgs2 expressions (Figure. 3A, B, C). Although the lower level of these gene expressions resulting from the addition of U0126 was not overcome by the addition of EGF, the addition of EGF to H89- or SB203580-containning medium overcame the negative effect of each inhibitor on these gene expressions (Figure. 3A, B, C).


Activation of PKA, p38 MAPK and ERK1/2 by gonadotropins in cumulus cells is critical for induction of EGF-like factor and TACE/ADAM17 gene expression during in vitro maturation of porcine COCs.

Yamashita Y, Hishinuma M, Shimada M - J Ovarian Res (2009)

Effect of H89, SB203580 or U0126 on expression of Has2(A), Tnfaip6(B) or Ptgs2(C) mRNA. For reference, the 0 h COC value was set as 1 and the data presented as the fold strength. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 10 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 10 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH, SB230580 and EGF for 10 h. The respective value of among Has2, Tnfaip6 and Ptgs2 mRNA were normalized according to those of β-actin mRNA to evaluate arbitrary units of the relative abundance of the targets. FSH(-): COCs were cultured without FSH and LH for 10 h; FSH(+): COCs were cultured with FSH and LH for 10 h; EGF(-): COCs were cultured without EGF for 10 h; EGF(+): COCs were cultured with EGF for 10 h; +H89: COCs were cultured with H89 for 10 h; +SB: COCs were cultured with SB203580 for 10 h; +U0126: COCs were cultured with U0126 for 10 h
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803446&req=5

Figure 3: Effect of H89, SB203580 or U0126 on expression of Has2(A), Tnfaip6(B) or Ptgs2(C) mRNA. For reference, the 0 h COC value was set as 1 and the data presented as the fold strength. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 10 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 10 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH, SB230580 and EGF for 10 h. The respective value of among Has2, Tnfaip6 and Ptgs2 mRNA were normalized according to those of β-actin mRNA to evaluate arbitrary units of the relative abundance of the targets. FSH(-): COCs were cultured without FSH and LH for 10 h; FSH(+): COCs were cultured with FSH and LH for 10 h; EGF(-): COCs were cultured without EGF for 10 h; EGF(+): COCs were cultured with EGF for 10 h; +H89: COCs were cultured with H89 for 10 h; +SB: COCs were cultured with SB203580 for 10 h; +U0126: COCs were cultured with U0126 for 10 h
Mentions: High levels of Has2, Tnfaip6 and Ptgs2 expression were observed in cumulus cells of COCs cultured for 10 h with FSH and LH as compared with those cultured without FSH and LH. The addition of H89, SB20580 or U0126 significantly suppressed FSH- and LH-induced Has2, Tnfaip6 and Ptgs2 expressions (Figure. 3A, B, C). Although the lower level of these gene expressions resulting from the addition of U0126 was not overcome by the addition of EGF, the addition of EGF to H89- or SB203580-containning medium overcame the negative effect of each inhibitor on these gene expressions (Figure. 3A, B, C).

Bottom Line: 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.The 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Veterinary Medicine, Faculty of Agriculture, Tottori University, 4-101 Koyamachou-minami, Tottori, 680-8553, Japan.

ABSTRACT

Objectives: During 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.

Methods: Areg, 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.

Results: When 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.

Conclusion: The 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.

No MeSH data available.