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

Journal of Ovarian Research 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 Yasuhisa Yamashita 1 Mitsugu Hishinuma 1 Masayuki Shimada 0 0 Graduate School of Biosphere Science, Hiroshima University , 1-4-4 Kagamiyama, Higashi-Hiroshima, 739-8528 , Japan 1 School of Veterinary Medicine, Faculty of Agriculture, Tottori University , 4-101 Koyamachou-minami, Tottori, 680-8553 , Japan 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 cumulusoocyte 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. - Background 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 induced 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 Tnfaip6 null mice [5] or Ptx3 null mice [7], number of ovulated oocytes decreased and in vivo fertilization was completely interrupted, suggesting that cumulus expansion was essential for both ovulation and fertilization processes. It is known that since LH receptor (Lhcgr) is mainly expressed in granulosa cells, the EGF-like factor produced in granulosa cells by LH surge acts on cumulus cells to induce cumulus expansion. Some factors were introduced to transmit the LH signal from granulosa cells to cumulus cells. For example, prostagrandin E2 (PGE2) that produced from granulosa cells and cumulus cells by prostagradin synthase 2 (PTGS2) was required for induction of Has2 and Tnfaip6 gene, cumulus expansion and oocyte meiotic resumption [8]. The EGF-like factors, Amphiregulin (AREG), Epiregulin (EREG) and -cellulin (BTC) were also recently reported as potent factor. The EGF-like factor was induced by LH stimuli in granulusa cells, and EGF receptor (EGFR) was localized on cumulus cells [9-11]. When mouse COCs were cultured with AREG, Has2, Tnfaip6 and Ptgs2 were expressed in cumulus cells. TACE/ ADAM17, the cleavage enzyme of EGF-like factor to soluble forms, was also expressed in porcine granulosa cells in vivo in response to hCG administration [12]. Thus, 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. In in vitro maturation of oocytes, COCs were recovered from antral follicles and cultured with FSH and/or LH. We previously showed that FSH and LH up-regulate EGF-like factor and Tace/Adam17 mRNA expression, and gonadotropins-induced cumulus expansion and oocyte maturation of porcine COCs were suppressed by EGF receptor tyrosine kinase inhibitor or TACE/ADAM17 inhibitor [13]. The results suggested that FSH- and LH-induced cumulus expansion was dependent on the expression and functions of EGF-like factors. Although the regulation of EGF-like factor expression in cancer cell lines has been reported [14,15], the mechanisms of EGF-like factor and TACE/ADAM17 expression in cumulus cells cultured with FSH and/or LH have remained unclear during in vitro maturation of porcine COCs. The binding of FSH and/or LH in granulosa cells to its own receptors led to rapidly and nongenomic activation of PKA, p38 MAPK, and PI3K in a cAMP-dependent manner [16] and of ERK1/2 via the SRC/RAS-dependent pathway [17]. In mice, since each inhibitor of PKA, p38 MAPK, PI3K or ERK1/2 suppressed the expression of cumulus expansion-related gene [10,18,19], cumulus expansion [18,19] or meiotic maturation of oocyte [20], we estimated that these signaling pathways induced by gonadotropin overlap the EGF-like factor-EGFR pathway, which induces full cumulus expansion and oocyte maturation. In this study, to clarify the intracellular pathway involved in EGF-like factor and Tace/Adam17 expression in cumulus cells, we examined the effect of PKA inhibitor (H89), p38 MAPK inhibitor (SB203580), PI3K inhibitor (LY294002) and MEK inhibitor (U0126) on Areg, Ereg and Tace/Adam17 expression in cumulus cells during in vitro maturation of porcine COCs. Additionally, we investigated the effect of these drugs on ERK1/2 phosphorylation, cumulus expansion and oocyte meiotic resumption in pig. Methods Materials High purified porcine FSH and porcine LH were gifts from the National Hormone and Pituitary Program (Rockville, MD, USA). Fetal calf serum (FCS) was obtained from Invitrogen (Carlsbad, CA, USA). Oligonucleotide poly- (dT) was purchased from Amersham Pharmacia Biotech (Newark, NJ, USA). Avian myeloblastosis virus reverse transcriptase and Taq DNA Polymerase were from Promega (Madison, WI). Routine chemicals and reagents were obtained from Nakarai Chemical Co. (Osaka, Japan) or Sigma (Sigma Chemical Co., St. Louis, MO, USA). In vitro culture of porcine COCs Isolation of porcine COCs was described previously [21]. Briefly, porcine ovaries were recovered from 5- to 7month-old prepubertal gilts at a local slaughterhouse. COCs were collected from the surfaces of intact healthy antral follicles measuring from 3 to 5 mm in (...truncated)