Zinc depletion activates porcine metaphase II oocytes independently of the protein kinase C pathway
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Zinc is an important trace element that regulates several biological functions. This study investigated the role of zinc in the metaphase (M) II arrest of porcine oocytes. N, N, N’, N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a Zn2+ chelator, was used to deplete free zinc from porcine MII stage oocytes. TPEN treatment significantly (P < 0.01) reduced the zinc content in the cytoplasm. The percentages of oocytes in which second polar body emission and pronuclear formation occurred increased as the concentration of TPEN increased (P < 0.01), and reached 93.64 ± 5.53% and 90.61 ± 9.10%, respectively, following treatment with 10 μM TPEN. Zinc depletion also resulted in cortical granule release and spindle depolarization. Maturation-promoting factor activity, as assessed by examining p34cdc2 activity, decreased (P < 0.05) following zinc depletion. Following TPEN treatment, embryos developed to the 4-cell stage but failed to reach the blastocyst stage. Zinc release is a common event in protein kinase C (PKC) activation. Therefore, we examined the impact of zinc depletion on phosphorylation of PKC substrates. Phosphorylation of PKC substrates was reduced (P < 0.05) in zinc-depleted oocytes, and this was rescued by phorbol 12-myristate 13-acetate (PMA) treatment. However, treatment of oocytes with both PMA and TPEN did not affect pronuclear formation or second polar body emission. These data are inconsistent with the hypothesis that oocyte activation caused by zinc depletion is mediated by the PKC pathway. This study shows that zinc has a novel role in maintaining MII arrest in porcine oocytes, but this is not mediated by the PKC pathway.
KeywordsOocyte Zinc Metaphase arrest PKC Activation
This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ00909801, PJ00956302 and PJ009594), Rural Development Administration, Republic of Korea.
TPEN didn’t affect Calcium content in oocytes. Oocytes were stained with fluro-4-AM and then been (A) activated by electronic pules as positive control. (B) 10 μM TPEN were injected into culture medium. Calcium content in oocytes after activation or TPEN treatment were recorded. (AVI 919 kb)
- Chou SS, Clegg MS, Momma TY, Niles BJ, Duffy JY, Daston GP, Keen CL (2004) Alterations in protein kinase C activity and processing during zinc-deficiency-induced cell death. Biochem J 383:63–71Google Scholar
- Colonna R, Clegg MS, Momma TY, Niles BJ, Duffy JY, Daston GP, Keen CL (1997) Protein kinase C is required for the disappearance of MPF upon artificial activation in mouse eggs. Mol Reprod Dev 48:292–299Google Scholar
- Kim AM, Vogt S, O'Halloran TV, Woodruff TK (2010) Zinc availability regulates exit from meiosis in maturing mammalian oocytes. Nat Chem Biol 6:674–681Google Scholar
- Kim AM, Bernhardt ML, Kong BY, Ahn RW, Vogt S, Woodruff TK, O'Halloran TV (2011) Zinc sparks are triggered by fertilization and facilitate cell cycle resumption in mammalian eggs. ACS Chem Biol 6:716–723Google Scholar
- Lisle RS, Anthony K, Randall MA, Diaz FJ (2013) Oocyte-cumulus cell interactions regulate free intracellular zinc in mouse oocytes. Reproduction. doi: 10.1530/REP-12-0338
- Mayes MA, Stogsdill PL, Prather RS (1995) Parthenogenic activation of pig oocytes by protein kinase inhibition. Biol Reprod 53:270–275Google Scholar
- Oh JS, Susor A, Schindler K, Schultz RM, Conti M (2013) Cdc25A activity is required for the metaphase II arrest in mouse oocytes. J Cell Sci 126:1081–1085Google Scholar
- Quan HM, Fan HY, Meng XQ, Huo LJ, Chen DY, Schatten H, Yang PM, Sun QY (2003) Effects of PKC activation on the meiotic maturation, fertilization and early embryonic development of mouse oocytes. Zygote 11:329–337Google Scholar
- Rickords LF, Peters MF, Stumpf TT (1992) Effect of the protein kinase C inhibitor staurosporine on oocyte activation of in vitro matured porcine oocytes. Biol Reprod 46:82Google Scholar
- Slater SJ, Kelly MB, Taddeo FJ, Rubin E, Stubbs CD (1994) Evidence for discrete diacylglycerol and phorbol ester activator sites on protein kinase C. Differences in effects of 1-alkanol inhibition, activation by phosphatidylethanolamine and calcium chelation. J Biol Chem 269:17160–17165Google Scholar