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, 20 (11), 1077-89

Maternally-derived Zinc Transporters ZIP6 and ZIP10 Drive the Mammalian Oocyte-To-Egg Transition

Affiliations

Maternally-derived Zinc Transporters ZIP6 and ZIP10 Drive the Mammalian Oocyte-To-Egg Transition

B Y Kong et al. Mol Hum Reprod.

Abstract

Rapid cellular zinc influx regulates early mammalian development during the oocyte-to-egg transition through modulation of the meiotic cell cycle. Despite the physiological necessity of this zinc influx, the molecular mechanisms that govern such accumulation are unknown. Here we show that the fully grown mammalian oocyte does not employ a transcriptionally based mechanism of zinc regulation involving metal response element-binding transcription factor-1 (MTF-1), as demonstrated by a lack of MTF-1 responsiveness to environmental zinc manipulation. Instead, the mammalian oocyte controls zinc uptake through two maternally derived and cortically distributed zinc transporters, ZIP6 and ZIP10. Targeted disruption of these transporters using several approaches during meiotic maturation perturbs the intracellular zinc quota and results in a cell cycle arrest at a telophase I-like state. This arrest phenocopies established models of zinc insufficiency during the oocyte-to-egg transition, indicating the essential function of these maternally expressed transporters. Labile zinc localizes to punctate cytoplasmic structures in the human oocyte, and ZIP6 and ZIP10 are enriched in the cortex. Altogether, we demonstrate a mechanism of metal regulation required for female gamete development that may be evolutionarily conserved.

Keywords: human; meiosis; oocyte; zinc; zinc transporters.

Figures

Figure 1
Figure 1
Zinc responsiveness is suppressed in meiotically competent oocytes. (A) Meiotically incompetent oocytes (non-surrounded nucleolus configuration, NSN) and meiotically competent oocytes (surrounded nucleolus configuration, SN) were isolated and cultured in control media for 14 h, and immunofluorescence done for metal response element-binding transcription factor-1 (MTF-1). Representative optical confocal sections in NSN (Ai) and SN (Aii) oocytes are shown. Dotted lines outline the nuclei. Representative images of NSN and SN nuclear configurations are shown as insets. Graph represents quantification of MTF-1 immunofluorescence intensity as a ratio of nuclear:cytoplasmic fluorescence intensity. (B) NSN oocytes were cultured in control media or media supplemented with 200 μM ZnSO4 (Zn) or 10 μM TPEN (TPEN) for 14 h. Representative transmitted light images of oocytes at end of culture are shown in i-iii; images of MTF-1 staining are shown in iv-vi, dotted lines outline the nuclei. MTF-1 immunofluorescence intensity was quantified as in (A). In (A) and (B), scale bars in transmitted light images = 80 μm, in confocal images = 25 μm. At least 20 oocytes were measured from each treatment group from three independent experiments. Data are shown as the mean ± SEM. Asterisks represent statistical significance when compared with NSN (A) or NSN control (B) at P < 0.001 as calculated by student t-test. (C) Quantitative real-time PCR (qRT–PCR) of MT1 and MT2 in NSN and SN oocytes at time of isolation (0 h) and at the end of culture in control, Zn or TPEN media as in (B). RNA was isolated from 50 oocytes from each treatment group in three separate experiments. Data are shown as the mean ± SEM. Asterisks in (Di, iii) represent statistical significance when compared with 0 h oocytes at P < 0.001 by student t-test. See also Supplementary data, Fig. S1.
Figure 2
Figure 2
Zip6 and Zip10 are highly expressed in the mouse oocyte and display a maternal expression pattern. (A–G) Relative amounts of zinc transporter mRNAs were evaluated across meiotic maturation and early embryo development by qRT–PCR. In each experiment, 50 oocytes or embryos were used to isolate mRNA, and the experiment was performed in triplicate. Data represent the mean ± SEM relative to the value obtained for PI oocytes. PI, prophase I; MII, metaphase II; 1C, 1-cell embryo; 2C, 2-cell embryo; 8C, 8-cell embryo; Blast, blastocyst embryo. (H) The relative amounts of each zinc transporter in the PI oocyte were analyzed. The data represent the mean ± SEM relative to the value obtained for ZnT1. Letters denote statistically significant differences between relative transcript abundance for each transporter (P < 0.01).
Figure 3
Figure 3
Temporal-spatial localization of ZIP6 and ZIP10 during meiotic maturation and preimplantation embryo development. (A) ZIP6 localization during meiotic maturation and early embryo development was determined by immunofluorescence using a custom synthesized antibody to ZIP6. Samples were collected at the indicated time points (NSN PI, SN PI, MI, MII, 2C, blastocyst) and processed for immunostaining. A minimum of 50 cells were examined in each group and representative images are shown. (B) ZIP10 localization was determined in a similar fashion as (A) with custom synthesis of an antibody to ZIP10. A minimum of 50 cells were examined in each group and representative images are shown. NSN, non-surrounded nucleolus; SN, surrounded nucleolus; PI, prophase I; MI, metaphase I; MII, metaphase II; 2C, 2-cell embryo. In merged images (Ai’-vi’, Bi’-iv’), ZIP6 (A) or ZIP10 (B) is green, F-actin is red and DNA is blue. Scale bars, 25 μm. At least 20 cells were visualized at each stage in three independent experiments. Asterisk represents an adjacent oocyte. See also Supplementary data, Fig. S2.
Figure 4
Figure 4
Disruption of Zip6 and Zip10 expression recapitulate phenotypes of TPEN-induced zinc insufficiency. (A) Oocytes were injected with 5 mM Zip6 or Zip10 morpholino (MO) and held in milrinone medium alone or milrinone medium supplemented with 10 μM U0126. Control oocytes were uninjected and held in milrinone medium. (B) Labile zinc distribution was visualized in injected oocytes by staining with 50 nM ZincBY-1. (C) At the end of culture, the percentage of oocytes undergoing germinal vesicle breakdown (GVBD) was calculated. Black bars represent oocytes held in milrinone, while white bars represent oocytes held in milrinone with U0126. (D–F) Oocytes were injected with 5 mM Zip6 or Zip10 MO, held for 10 h in milrinone medium and then transferred to in vitro maturation medium for 14 h before being imaged for labile zinc distribution (E) or fixed for immunostaining (F). (F) Spindle structure was interrogated by labeling oocytes with α-tubulin (green), actin (red) and chromatin (blue). Representative confocal images of MII and telophase I-like spindles are shown as Z-stack projections. (G) The percentage of oocytes arresting in a telophase I-like phenotype was calculated for control uninjected and Zip6 and Zip10 MO injected oocytes. Graphical data are presented as the mean ± SEM of three independent trials with at least 30 oocytes per trial. Statistical differences were calculated according to one-way ANOVA with Bonferroni post hoc test in comparison to the Control group (P < 0.001). Scale bar, 25 μm.
Figure 5
Figure 5
Neutralization of ZIP6 and ZIP10 function by antibody incubation during meiosis causes telophase I-like arrest. (A) Oocytes were cultured in in vitro maturation (IVM) medium with ZIP6 and/or ZIP10 antibody for 14 h. Base IVM medium and IVM medium with rabbit IgG antibody served as controls. At the end of culture, all samples were fixed and labeled for α-tubulin (green), F-actin (red) and chromatin (blue) to interrogate spindle morphology. Representative confocal images of control MII spindles (i) and telophase I-like spindles (two chromatin masses with midbody, ii; two chromatin masses without midbody, iii; two chromatin masses with cytokinesis failure, iv) are shown as Z-stack projections. Scale bars = 25 μm. (B) The percentage of oocytes arresting in a telophase I-like phenotype was calculated. Data are presented as the mean ± SEM of three independent trials with at least 30 oocytes per trial. Statistical differences were calculated according to one-way ANOVA with Bonferroni post hoc test in comparison to controls (P < 0.001).
Figure 6
Figure 6
Labile zinc, ZIP6 and ZIP10 are conserved in the human oocyte. (A) Labile zinc was visualized in human oocytes using two zinc-specific fluorophores: ZincBY-1 (left) and FluoZin-3 AM (right). For each oocyte, a transmitted light image, an optical section and a projection are shown. (B) ZIP6 and ZIP10 were visualized using specific antibodies. A single optical section of each oocyte is shown. A total of 13 human oocytes from 6 individuals were used to perform these experiments as described in Table II. Representative images are shown. Labile zinc = green, DNA = blue, and zinc transporters = grayscale. Scale bars, 50 mm.

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