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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 rate of oocyte exhibiting GVBD. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 20 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 20 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH, SB230580 and EGF for 20 h. FSH(-): COCs were cultured without FSH and LH for 20 h; FSH(+): COCs were cultured with FSH and LH for 20 h; EGF(-): COCs were cultured without EGF for 20 h; EGF(+): COCs were cultured with EGF for 20 h; +H89: COCs were cultured with H89 for 20 h; +SB: COCs were cultured with SB203580 for 20 h; +U0126: COCs were cultured with U0126 for 20 h
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Figure 4: Effect of H89, SB203580 or U0126 on rate of oocyte exhibiting GVBD. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 20 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 20 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH, SB230580 and EGF for 20 h. FSH(-): COCs were cultured without FSH and LH for 20 h; FSH(+): COCs were cultured with FSH and LH for 20 h; EGF(-): COCs were cultured without EGF for 20 h; EGF(+): COCs were cultured with EGF for 20 h; +H89: COCs were cultured with H89 for 20 h; +SB: COCs were cultured with SB203580 for 20 h; +U0126: COCs were cultured with U0126 for 20 h

Mentions: When COCs were cultured without FSH and LH for 20 h, the proportion of oocytes exhibiting GVBD was less than 25%. FSH and LH significantly increased the proportion of oocytes exhibiting GVBD. This higher rate was significantly decreased by H89 or SB20580, and the suppression was counteracted by the addition of EGF (Figure 4). Treatment with U0126 also significantly suppressed the GVBD rate, whereas the addition of EGF did not overcome the negative effects.


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 rate of oocyte exhibiting GVBD. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 20 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 20 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH, SB230580 and EGF for 20 h. FSH(-): COCs were cultured without FSH and LH for 20 h; FSH(+): COCs were cultured with FSH and LH for 20 h; EGF(-): COCs were cultured without EGF for 20 h; EGF(+): COCs were cultured with EGF for 20 h; +H89: COCs were cultured with H89 for 20 h; +SB: COCs were cultured with SB203580 for 20 h; +U0126: COCs were cultured with U0126 for 20 h
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effect of H89, SB203580 or U0126 on rate of oocyte exhibiting GVBD. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 20 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 20 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH, SB230580 and EGF for 20 h. FSH(-): COCs were cultured without FSH and LH for 20 h; FSH(+): COCs were cultured with FSH and LH for 20 h; EGF(-): COCs were cultured without EGF for 20 h; EGF(+): COCs were cultured with EGF for 20 h; +H89: COCs were cultured with H89 for 20 h; +SB: COCs were cultured with SB203580 for 20 h; +U0126: COCs were cultured with U0126 for 20 h
Mentions: When COCs were cultured without FSH and LH for 20 h, the proportion of oocytes exhibiting GVBD was less than 25%. FSH and LH significantly increased the proportion of oocytes exhibiting GVBD. This higher rate was significantly decreased by H89 or SB20580, and the suppression was counteracted by the addition of EGF (Figure 4). Treatment with U0126 also significantly suppressed the GVBD rate, whereas the addition of EGF did not overcome the negative effects.

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.