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. 2012 Feb;153(2):873-86.
doi: 10.1210/en.2011-1599. Epub 2011 Dec 6.

Zinc depletion causes multiple defects in ovarian function during the periovulatory period in mice

X Tian  1 , F J Diaz
Affiliations

Zinc depletion causes multiple defects in ovarian function during the periovulatory period in mice

X Tian et al. Endocrinology. 2012 Feb.

Abstract

Shortly before ovulation, the oocyte acquires developmental competence and granulosa cells undergo tremendous changes including cumulus expansion and luteinization. Zinc is emerging as a key regulator of meiosis in vitro, but a complete understanding of zinc-mediated effects during the periovulatory period is lacking. The present study uncovers the previously unknown role of zinc in maintaining meiotic arrest before ovulation. A zinc chelator [N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN)] caused premature germinal vesicle breakdown and associated spindle defects in denuded oocytes even in the presence of a phosphodiesterase 3A inhibitor (milrinone). TPEN also potently blocked cumulus expansion by blocking induction of expansion-related transcripts Has2, Ptx3, Ptgs2, and Tnfaip6 mRNA. Both meiotic arrest and cumulus expansion were rescued by exogenous zinc. Lack of cumulus expansion is due to an almost complete suppression of phospho-Sma- and Mad-related protein 2/3 signaling. Consistent with a decrease in phospho-Sma- and Mad-related protein 2/3 signaling, TPEN also decreased cumulus transcripts (Ar and Slc38a3) and caused a surprising increase in mural transcripts (Lhcgr and Cyp11a1) in cumulus cells. In vivo, feeding a zinc-deficient diet for 10 d completely blocked ovulation and compromised cumulus expansion. However, 42.5% of oocytes had prematurely resumed meiosis before human chorionic gonadotropin injection, underscoring the importance of zinc before ovulation. A more acute 3-d treatment with a zinc-deficient diet did not block ovulation but did increase the number of oocytes trapped in luteinizing follicles. Moreover, 23% of ovulated oocytes did not reach metaphase II due to severe spindle defects. Thus, acute zinc deficiency causes profound defects during the periovulatory period with consequences for oocyte maturation, cumulus expansion, and ovulation.

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Figures

Fig. 1.
Fig. 1.
A–C, Bright-field images of oocytes cultured for 10, 14, 18, and 20 h in medium containing 10 μm milrinone (A), milrinone (Mil) plus 10 μm TPEN (B), or milrinone (Mil), TPEN, and 10 μm zinc (Zn) (C). Scale bar, 100 μm. D, Proportion of oocytes undergoing GVBD in milrinone (M) alone, milrinone plus TPEN (MT), or milrinone, TPEN, and 10 μm zinc (MTZ). Values are mean ± sem. a–c, Significant difference by ANOVA and Tukey's post hoc test, P < 0.05; n = 4.
Fig. 2.
Fig. 2.
A, Spindle staining using tubulin antibodies to stain microtubules, phalloidin-TRITC to stain actin filaments, and DAPI to stain DNA in oocytes cultured in milrinone for 20 h (control). B, Spindle staining of oocytes cultured with milrinone and TPEN (10 μm) for 20 h. C, Proportion of oocytes undergoing GVBD (germinal vesicle breakdown) from mice fed a control diet or a ZDD (zinc deficient diet) for 10 d and primed with PMSG (but not hCG) on d 8. D, Spindle staining of oocytes from mice fed control diet. E, Spindle staining of oocytes from animals fed a ZDD for 10 d. F, Levels of cAMP in oocytes treated with milrinone or milrinone plus TPEN and cultured for 6 and 10 h. G, MPF activity in oocytes treated with milrinone (M), milrinone and TPEN (MT), or milrinone, TPEN and zinc (10 μm) (MTZ) for 6, 10, and14 h. MPF activity in GV and MII oocytes are provided as a reference. H, Immunostaining for H3K9Ac in control and TPEN-treated oocytes cultured for 20 h. I, Immunostaining for H3K4me3 in control and TPEN-treated oocytes cultured for 20 h. J, Negative control with secondary antibody only. Scale bars, 10 μm (A, B, D, and E) and 50 μm (H–J). Values are mean ± sem. a–c, Significant differences by ANOVA followed by Tukey's post hoc test, P < 0.05; n = 3. *, Significant difference by Student's t test, P < 0.05; n = 3.
Fig. 3.
Fig. 3.
Panel A, Cumulus expansion in COC treated with medium only (CON), EGF (10 ng/ml), TPEN (10 μm), EGF plus TPEN (ET), or EGF, TPEN, and zinc (ETZ) (10 μm) for 15 h. Scale bar, 100 μm. Panel B, Levels of expansion-related transcripts Has2, Tnfaip6, Ptx3, and Ptgs2 in COC cultured as in panel A for 6 h. Panel C, Levels of expansion-related transcripts Has2, Tnfaip6, Ptx3, and Ptgs2 in COC cultured in control (CON), EGF, TPEN, and EGF plus TPEN for 10 h. D, Western blot for pSMAD2 (upper band) and actin (ACTB, lower band) in control (C) and TPEN-treated (T) COC for 6 h. E, Western blot for pMAPK3/1 in COC treated with medium only (C, control), EGF (E, 10 ng/ml), EGF plus TPEN (E+T), or TPEN (T, 10 μm) for 4 h. F, Levels of cumulus marker transcripts (Ar, Amh, and Slc38a3) in control and TPEN-treated COC cultured for 20 h. G, Levels of mural marker transcripts (Cyp11a1 and Lhcgr) in control and TPEN-treated COC cultured for 20 h. Values are mean ± sem. a–c, Significant differences by ANOVA followed by Tukey's post hoc test, P < 0.05; n = 4. *, Significant difference by Student's t test, P < 0.05; n = 3–4.
Fig. 4.
Fig. 4.
A, Number of ovulated oocytes from mice fed a control diet or a ZDD for 10 d. Animals were primed with PMSG on d 8 and hCG on d 10, and ovulated COC were collected 13 h later. B, Hematoxylin- and eosin-stained sections from animals fed a control diet. Scale bar, 500 μm. C, Hematoxylin- and eosin-stained sections from animals fed a ZDD diet. Scale bar, 500 μm. D and E, Close-up of two large antral follicles from ZDD animals that failed to ovulate. Scale bar, 100 μm. F, Close-up of a COC from a ZDD animal that failed to undergo cumulus expansion, but the oocyte still matured as indicated by condensed chromosomes. Scale bar, 50 μm. Values are mean ± sem. *, Significant difference by Student's t test, P < 0.05; n = 5–6.
Fig. 5.
Fig. 5.
A, Number of ovulated oocytes from mice fed a control diet or a ZDD for 3 d and primed with PMSG on d 1 and hCG on d 3 and collected 13 h later. B, Hematoxylin- and eosin-stained sections from animals fed a control diet. Scale bar, 500 μm. C, Hematoxylin- and eosin-stained sections from animals fed a ZDD diet. Scale bar, 500 μm. D–F, Close-up of three large luteinizing follicles on the same ovary with trapped oocytes. Scale bar, 100 μm. G, Proportion of ovulated oocytes with a polar body from animals fed control or a ZDD for 3 d. H, Spindle staining of ovulated oocytes from control animals stained with antitubulin, phalloidin-TRITC, and DAPI to localize microtubules, actin filaments, and DNA, respectively. Scale bar, 50 μm. I, Spindle staining of ovulated oocytes from animals fed a ZDD showing examples of defective maturation in oocytes lacking a polar body (33% of total). Scale bar, 50 μm. Arrowheads indicate location of attached cumulus cells. Values are mean ± sem. *, Significant difference by Student's t test, P < 0.05; n = 4–6.
Fig. 6.
Fig. 6.
Model showing proposed action of zinc-dependent mechanisms in oocytes and cumulus cells before and during ovulation. Before ovulation, the combined effects of natriuretic peptide precursor C (NPPC) and GPR3 and GPR12 maintain high cAMP to prevent MPF activation. NPPC binds to the natriuretic peptide receptor 2 (NPR2) receptor and generates cGMP, which passes through gap junctions into the oocyte and inhibits PDE3A. GPR3 and GPR12 activate adenylate cyclase (AC) to generate cAMP in the oocyte. Zinc enters the oocyte and cumulus cells through specific transporters. In oocytes, zinc may negatively regulate cAMP levels by inhibiting adenylate cyclase and stimulating PDE activity. Additionally, zinc is involved in preventing meiotic resumption by regulating MPF or downstream processes to prevent premature GVBD. In cumulus cells, zinc is required to enable pSMAD2/3 signaling to maintain the cumulus phenotype. After the LH surge, EGF-LP in conjunction with zinc-enabled pSMAD2/3 signaling promotes cumulus expansion. In oocytes, a zinc-mediated process is required for completion of meiosis I.
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References

    1. Zhang M, Su YQ, Sugiura K, Xia G, Eppig JJ. 2010. Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes. Science 330:366–369 - PMC - PubMed
    1. Vaccari S, Weeks JL, 2nd, Hsieh M, Menniti FS, Conti M. 2009. Cyclic GMP signaling is involved in the luteinizing hormone-dependent meiotic maturation of mouse oocytes. Biol Reprod 81:595–604 - PMC - PubMed
    1. Norris RP, Ratzan WJ, Freudzon M, Mehlmann LM, Krall J, Movsesian MA, Wang H, Ke H, Nikolaev VO, Jaffe LA. 2009. Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte. Development 136:1869–1878 - PMC - PubMed
    1. Mehlmann LM, Saeki Y, Tanaka S, Brennan TJ, Evsikov AV, Pendola FL, Knowles BB, Eppig JJ, Jaffe LA. 2004. The Gs-linked receptor GPR3 maintains meiotic arrest in mammalian oocytes. Science 306:1947–1950 - PubMed
    1. Hinckley M, Vaccari S, Horner K, Chen R, Conti M. 2005. The G-protein-coupled receptors GPR3 and GPR12 are involved in cAMP signaling and maintenance of meiotic arrest in rodent oocytes. Dev Biol 287:249–261 - PubMed

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