Study Of PKC δ/θ Signaling Mediating FSH-induced Mouse Oocyte Meiotic Resumption

The mammalian oocytes maturation is a dynamic process of cell cycle transition in oocytes induced by external signal. It is reported that Protein kinase C (PKC) signaling plays an important role in gonadotropin-induced oocyte meiotic resumption and epidermal growth factor (EGF)-like factors mediate gonadotropin-induced rodent oocyte maturation via EGF receptor EGFR. The early studies in our lab suggested that PKC may activate EGF/EGFR systems possibly by shed EGF and/or EGF-like factors, not by transcription-dependent mechanism in gonadotropin-induced oocyte maturation. It is proved that tumor necrosis factor-a-converting enzyme (TACE) is principally responsible for the shedding of EGF-like factors which bind to and activate EGFR signaling.In our study, by using mouse cumulus cell enclosed oocytes (CEOs) cultured model, we investigated the activation mechanism of TACE induced by PKC signaling in FSH-induced mouse oocyte meiotic maturation.First of all, the present investigation was undertaken to examine the effect of different PKC isoforms on FSH-induced mouse CEOs meiotic resumption. We cultured mouse CEOs with PKC a and PKC β1special inhibitor Go6976, PKC δ and PKC θ inhibitor rottlerin (MTX), and PKC ζ Pseudosubstrate Inhibitor (Pi), respectively. The results showed that Go6976and PKC ζ Pseudosubstrate Inhibitor (Pi) had no effect on FSH-induced mouse meiotic resumption whereas the effect of FSH on mouse oocyte maturation was dose-dependently inhibited by MTX. Furthermore, the addition of100ng/ml Amphiregulin (AR) to the culture containing MTX could reverse the suppressive effects of the inhibitor. In addition, the expression of transcripts encoding EGF-like factors Areg, Ereg, and Btc mRNA levels in CEOs was dramatically increased after4h during the CEOs cultured with FSH treatment. However, the high level of mRNA expression of these genes was not affected by treatment with MTX. These findings suggest that PKC δ/θ participate in FSH-induced oocyte maturation, but not by stimulating the mRNA expression of Amphiregulin (AR), epiregulin (ER) and betacellulin (Btc).Then, we investigated whether TACE was involved in FSH-induced mouse oocyte meiotic resumption. The expression of Tace mRNA level and TACE protein level was significantly increased after FSH treatment in FSH-induced mouse CEOs maturation. Besides, FSH-induced CEOs meiotic resumption was dose-dependently inhibited by TACE selective inhibitor TAPI-2. These results indicate that TACE is involved in FSH-induced mouse CEOs meiotic resumption.Finally, we determined the mechanism of PKC δ/θ signaling regulate the activity of TACE in FSH-induced mouse oocyte meiotic resumption. The results showed that the effect of FSH on oocyte maturation was blocked by diphenyleneiodonium chloride (DPI)(a special inhibitor of NADPH Oxidase) and1,3-dimethyl-2-thiourea (DMTU)(ROS scavenger) in a dose-dependent manner, respectively, which could be partially rescued by Amphiregulin (AR). Then in our immunofluorescence experiments, we found that FSH could prompt the cytosolic components of NADPH Oxidase (NOX) p47phox and p67phox transferring to the plasma membrane to form the complete enzyme NADPH Oxidase in cumulus cells in1h culture compared with control. But the translocation of p47phox and p67phox could be prevented by rottlerin in cumulus cells. In addition, PKC activator phorbol12-myristate13-acetate (PMA) could mimic FSH action on mouse oocyte maturation, whereas DPI significantly suppressed PMA-induced mouse oocyte meiotic resumption. Additionally, the study showed that TACE activity was notably increased at2h of culture after FSH treatment as compared with that in CEOs before culture. However, the high level of TACE activity was markedly suppressed when CEOs cultured in the presence of DPI and DMTU respectively compared with control. The inhibition of these inhibitors on TACE activity was more significant after4h. These results suggeste that PKC δ/θ may mediate NADPH Oxidase by regulating the translocation of p47phox and p67phox, and the R.OS derived from NADPH Oxidase activate TACE for the shedding of EGF-like factors.In summary, PKC δ/θ signaling is necessary for FSH-induced mouse oocyte meiotic resumption. All these results indicate that TACE plays a critical role in FSH-induced meiotic resumption of mouse oocytes, and TACE activity in cumulus cells is mediated by PKC δ/θ signaling, which suggesting that PKC δ/θ-NOX-ROS-TACE cascades paticipate in the shedding EGF and/or EGF-like factors to activate EGFR, during FSH-induced mouse CEOs meiotic resumption in vitro.