CROSS-REFERENCE TO RELATED APPLICATIONS
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The present invention claims the benefit of U.S. Provisional Patent Application Ser. No. 61/475,472, filed Apr. 14, 2011, which is incorporated by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under Grant Nos. HD021921 and GM038784 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD OF THE INVENTION
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The invention relates generally to compositions and methods for the detection of zinc. In particular, compositions and methods are provided to detect physiological changes in zinc concentration (e.g., extracellular, intracellular, etc.) and to correlate them to cellular phenomena.
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OF THE INVENTION
The biological functions of metal ions have traditionally been thought to be limited to structural and catalytic roles within proteins. However, metal ions are also known to play signaling roles at the cellular level. For example, the alkaline earth metal calcium is one metal in which biological signaling roles are particularly well-established. Periodic elevations in the intracellular concentration of free calcium ions, also known as calcium transients or oscillations, are readily detected using fluorescent probes and are known to drive a number of biological processes (Berridge et al. (2003) Nat Rev Mol Cell Biol 4, 517-29.; herein incorporated by reference in its entirety). This is perhaps best illustrated in the egg, where repetitive calcium transients are among the earliest observable events after fertilization (Lawrence et al. (1997) Development 124, 233-41.; herein incorporated by reference in its entirety). Several parameters of these calcium transients, including total number, frequency, and amplitude influence which downstream developmental events are initiated (Ozil et al. (2006) Dev Biol 300, 534-44.; Ducibella et al. (2002) Dev Biol 250, 280-91.; Toth et al. (2006) Reproduction 131, 27-34.; herein incorporated by reference in their entireties). These cellular processes, which include cortical granule (CG) exocytosis and cell cycle progression can be initiated in the absence of sperm by stimulatory agents that induce parthenogenesis (Kline & Kline. (1992) Dev Biol 149, 80-9.; Tahara et al. (1996) Am J Physiol 270, C1354-61.; Liu & Maller. (2005) Curr Biol 15, 1458-68.; Madgwick et al. (2006) J Cell Biol 174, 791-801.; Battaglia & Gaddum-Rosse. (1987) Gamete Res 18, 141-52.; Ozil. (1990) Development 109, 117-27.; Zhang et al. (2005) Hum Reprod 20, 3053-61.; Tingen (2010) Science 330, 453; herein incorporated by reference in their entireties). Interestingly, the normal pattern of calcium oscillations is disrupted in eggs matured under conditions which limit the availability of the transition metal zinc (Kim et al. (2010) Nat Chem Biol 6, 674-81.; herein incorporated by reference in its entirety), suggesting that the physiologies of these metals are somehow connected in the egg.
In a departure from its well-established role as an enzymatic cofactor or structure stabilizing agent, fluctuations in the total concentration of intracellular zinc have recently been shown to contribute to the proper cell cycle regulation in maturing oocytes. Intracellular zinc levels increase by more than fifty percent and over 1010 ions per cell are accrued during the final stage of oocyte maturation, also known as meiotic maturation (Kim et al. (2010) Zinc availability regulates exit from meiosis in maturing mammalian oocytes, Nat Chem Biol 6, 674-81.; herein incorporated by reference in its entirety). This significant cellular metal accumulation event occurs over a remarkably short time interval and is a physiological imperative, as insufficient accumulation of zinc leads to a premature meiotic arrest at telophase I instead of metaphase II (Kim et al. (2010) Nat Chem Biol 6, 674-81.). This zinc-dependent meiotic checkpoint arises, in part, because zinc-insufficient eggs fail to reestablish maturation promoting factor (MPF) activity (Bernhardt et al. (2010) Biol Reprod, published ahead of print Nov. 10, 2010.; herein incorporated by reference in its entirety), which is necessary for eggs to set up and maintain meiotic arrest at metaphase II.
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OF THE INVENTION
In some embodiments, the present invention provides a Zn-responsive probe comprising: (a) a Zn-binding group configured to coordinate one or more Zn ions; (b) a signaling moiety configured to emit a detectable signal; and (c) an attachment group, wherein said attachment group is a chemically reactive with one or more functional groups. In some embodiments, the detectable signal of the signaling moiety is altered by coordination of one or more Zn ions by the Zn-binding moiety. In some embodiments, the signaling moiety comprises a fluorophore. In some embodiments, the fluorescence intensity, emission spectra, and/or excitation spectra of the signaling moiety is altered by coordination of one or more Zn ions by the Zn-binding moiety. In some embodiments, the interaction of the attachment group with a chemically reactive functional group results in covalent or stable non-covalent attachment of the Zn-responsive probe to the functional group. In some embodiments, the Zn-binding group is attached to the signaling group, and the signaling group is attached to the attachment group. In some embodiments, the one or more attachments are though a linker group. In some embodiments, the functional group is attached to a surface.
In some embodiments, the present invention provides a method of non-invasively detecting Zn release and/or uptake by a cell or cells comprising: (a) contacting a sample comprising a cell or cells with a Zn-responsive probe (e.g., a Zn-responsive probe attached to a solid surface); (b) detecting the signal emitted by the signaling moiety over time; and (c) correlating signal emitted with Zn concentration (e.g., intracellular or extracellular). In some embodiments, the sample comprises one or more oocytes (e.g., human oocytes (e.g., for use in in vitro fertilization), non-human oocyctes (e.g., oocytes of a non-human primate, domestic animal (e.g., feline, canine, etc.), agricultural animal (e.g., bovine, porcine, etc.)). In some embodiments, the present invention further comprises (d) removing the sample from the Zn-responsive probe (e.g, separating the cell and the Zn-responsive probe). In some embodiments, removing the sample from the Zn-responsive probe (or the Zn-responsive probe from the sample) is facilitated by the attachment of the probe to a solid surface.
In some embodiments, the present invention provides a method of detecting the fertilization of an oocyte comprising: (a) contacting a sample comprising an metaphase-II-arrested oocyte with a Zn-responsive probe (e.g., a Zn-responsive probe attached to a solid surface); (b) contacting the sample with sperm; (c) detecting a significant increase in extracellular Zn concentration based on a change in the signal of the Zn-responsive probe; and (d) correlating the significant increase in extracellular Zn concentration to fertilization of the oocyte by the sperm. In some embodiments, methods further comprise (f) removing the oocyte from the Zn-responsive probe; and (g) implanting the fertilized oocyte into a subject. In some embodiments, the Zn-responsive probe is attached to a surface. In some embodiments, removing the oocyte from the Zn-responsive probe is facilitated by the attachment of the probe to a surface. In some embodiments, attachment of the Zn-responsive probe to a surface prevents uptake of the probe into the oocyte.
In some embodiments, the present invention provides a method of evaluating the quality of an oocyte for fertilization comprising: (a) contacting a surface with a sample comprising the oocyte, wherein said surface displays a plurality of Zn-responsive probes (e.g., Zn-responsive probes are attached to the surface); (b) detecting the signal emitted by the Zn-responsive probes over time; (c) correlating the signal emitted with extracellular Zn concentration, wherein a decrease in extracellular Zn is indicative of an oocyte that is ready for fertilization. In some embodiments, the oocyte is in prophase-1-arrest upon contacting with the surface. In some embodiments, methods further comprise (d) removing the oocyte from the Zn-responsive probe. In some embodiments, removing the oocyte from the Zn-responsive probe is facilitated by the attachment of the probe to a surface. In some embodiments, attachment of the Zn-responsive probe to a surface prevents uptake of the probe into the oocyte.
In some embodiments, the present invention provides compositions comprising a solid surface displaying one or more Zn-responsive probes, wherein said Zn-responsive probes comprise: (a) a Zn-binding group configured to coordinate one or more Zn ions; and (b) a signaling moiety configured to emit a detectable signal. In some embodiments, the detectable signal of the signaling moiety is altered by coordination of one or more Zn ions by the Zn-binding moiety. In some embodiments, the signaling moiety comprises a fluorophore. In some embodiments, the fluorescence intensity, emission spectrum, and/or excitation spectrum of the signaling moiety is altered by coordination of one or more Zn ions by the Zn-binding moiety. In some embodiments, the composition further comprises a linker. In some embodiments, the linker connects the Zn-binding group with the signaling moiety. In some embodiments, the linker connects the Zn-binding group and/or the signaling moiety to the solid surface. In some embodiments, the Zn-responsive probes are attached to the solid surface through the interaction of a attachment group on the Zn-responsive probe and an anchor moiety on the solid surface. In some embodiments, the solid surface is selected from the group comprising: a plate, bead, well, slide, biopolymer, cell surface, and tube.
In some embodiments, the present invention provides a method for detection of Zn in a sample (e.g., by any suitable method). In some embodiments, a change in Zn concentration is detected. In some embodiment, Zn is detected by a Zn-responsive probe. In some embodiments, Zn is detected without a probe. In some embodiments, detection of Zn is correlated to a biological function or process (e.g., oocyte fertilization). In some embodiments, a Zn-responsive probe is attached to a solid surface (e.g., bead, plate, slide, well, etc.). In some embodiments, attachment of a Zn-responsive probe to a solid surface facilitates removal of the probe from a sample without contaminating the sample with Zn-responsive probe. In some embodiments, attachment of a Zn-responsive probe to a solid surface facilitates removal of the sample from a surface (e.g., (tube, well, slide, etc.) without contaminating the sample with Zn-responsive probe. In some embodiments, attachment of the Zn-responsive probe to a surface prevents uptake of the probe into the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
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The description provided herein is better understood when read in conjunction with the accompanying drawings, which are included by way of example and not by way of limitation.
FIG. 1 shows images depicting how changes in extracellular zinc concentration are readily monitored with FluoZin-3 during in vitro fertilization. Rapid, repetitive increases in fluorescence intensity were detected (a) by ROI analysis (denoted by white boxes in b-d). These spikes in fluorescent intensity involve the coordinated release of zinc from the cell in a phenomenon defined as a “zinc spark.” Each zinc spark can be distinguished in the time-lapse series (b, 00:25:12) against background fluorescence (representative example in c, 01:32:56). Successful fertilization was confirmed by the extrusion of a second polar body (d). Zinc sparks were also noted during strontium chloride-induced parthenogenesis (e, 00:02:56).
FIG. 2 shows zinc sparks observed in eggs from two different non-human primate species, Macaca mulatta (a, b) and Macaca fascicularis (c, d). In both cases, a single calcium transient was induced by ionomycin. Intracellular calcium was monitored with Calcium Green-1 AM, and extracellular zinc with FluoZin-3. The first panel begins at 00:04:35 in c and 00:06:37 in d. Each subsequent panel represents an image acquired 4 s following the previous panel. Time is expressed as hh:mm:ss, wherein 00:00:00 represents the start of image acquisition. Scale bar=40 μm in c and d.
FIG. 3 shows that zinc sparks are polarized and are immediately preceded by intracellular calcium transients. Shortly following activation, zinc sparks occur around the egg cortex with the exception of a zinc spark-free region (a, 00:21:04, arrowhead). This spark-free region corresponds to the region containing the meiotic spindle, where the second polar body is extruded (a, 01:20:00, arrowhead). Egg activation was confirmed by simultaneously monitoring intracellular calcium oscillations with Calcium Green-1 AM every 4 s (b). Intracellular calcium increases immediately before a zinc spark, as evident when images were collected at a faster acquisition rate of every 100 ms in an independent experiment (c). Time is expressed as hh:mm:ss, wherein 00:00:00 represents the start of image acquisition. In all cases, extracellular zinc was detected with FluoZin-3.
FIG. 4 shows that zinc sparks did not occur in the absence of calcium transients. The membrane permeable derivative of FluoZin-3 (FluoZin-3 AM) did not detect intracellular oscillations in zinc (a), suggesting that those detected by Calcium Green-1 AM were specific to calcium. Insets are brightfield images taken at the beginning (left, first polar body denoted as PB1) and end (right) of the acquisition period. In each case, the presence of the second polar body (PB2) at the conclusion of imaging was observed, confirming successful egg activation and development. Zinc sparks were absent in the absence of an activating agent such as strontium chloride (b), or in eggs treated with the calcium-selective chelator BAPTA AM (c). Representative images illustrate that BAPTA AM-treated eggs neither extrude a second polar body (b, 2 hrs postactivation, or hpf) nor form a pronucleus (c, 6 hpf). Intracellular calcium was detected by Calcium Green-1 AM (black line) and extracellular zinc was detected by FluoZin-3 (gray line) in b and c. Scale bar=75 μm in d and e.
FIG. 5 shows images depicting cortically polarized zinc ions in the mouse egg. Total zinc, as detected by synchrotron-based x-ray fluorescence (a, b), is uniquely polarized in the unfertilized egg (a; i-iii represent replicates). This distribution is absent in the other essential transition elements, such as iron (b; i-iii represent replicates). The range of each group of images is given units of μg/cm2. Labile (chelator-accessible) zinc, as detected by confocal microscopy (c-h), also has a hemispherical distribution in the live egg, as detected by two chemically distinct zinc fluorophores: zinquin ethyl ester (c-e) and FluoZin-3 AM (f-h). Co-staining with a DNA marker (Syto 64 in d, Hoechst 33342 in g) revealed that zinc was concentrated at the vegetal pole away from the meiotic spindle.
FIG. 6 shows total zinc content of the egg gradually decreases upon fertilization. Eggs and embryos were analyzed by XFM for their transition metal content. The mean zinc quota was highest in the in vivo ovulated (IVO) MII egg and trended towards a decrease following fertilization (see 2 and 6 hpf), reaching its lowest mean abundance in the two-cell embryo. The iron and copper quotas were an order of magnitude less than zinc at all time points examined.
FIG. 7 shows intracellular zinc decreases in the activated egg. Corresponding to the onset of the zinc sparks (FluoZin-3, gray line), there is a gradual decrease in intracellular labile zinc that is detectable with a membrane-permeable zinc fluorophore (FluoZin-3 AM, black line). Successful egg activation and development was confirmed using simultaneous brightfield imaging, which revealed extrusion of a second polar body (PB2). The first (0 min, left) and final (30 min, right) brightfield images are shown.
FIG. 8 shows optical sections from areas near the meiotic spindle (as detected by DNA markers Syto 64 and Hoechst 33342), selected from the complete confocal Z-series and projected to show the cortical localization of zinc-enriched vesicles as detected by two independent fluorophores, zinquin ethyl ester and FluoZin-3 AM. Merged images are also shown for clarity.
FIG. 9 shows exocytosis was inhibited by treating eggs with cytochalasin B prior to activation with strontium chloride. Cytochalasin B did not affect the calcium oscillations (a) but blocked all but the first zinc spark in most cases (b).
FIG. 10 shows dose response of unfertilized eggs to increasing concentrations of zinc pyrithione (ZnPT). Unfertilized eggs were treated with 10, 20, or 50 μM ZnPT for 15 min. A clear cytotoxic phenotype was only seen at 50 μM concentration. Thus, the effects of pyrithione are significantly below the threshold concentration (i.e., 500% lower) and do not involve cytotoxicity, as pyrithione was used at a minimum concentration (10 μM) and for a shorter duration (10 min).
FIG. 11 shows sustained elevation of intracellular zinc availability following egg activation leads to reestablishment of metaphase arrest. Eggs were activated with strontium chloride (SrCl2) then treated with zinc pyrithione (ZnPT) 1.5 hours later (a). At 6 h post-activation, control eggs form pronuclei (b, arrowheads) whereas the majority of eggs treated with ZnPT do not (c). When visualized by fluorescence, control eggs display decondensed DNA organized within a defined nucleus (d). F-actin is homogeneous around the egg\'s cortex (e) and α-tubulin remains organized as a spindle midbody remnant (f), which is visible between the egg and the second polar body (g, merged image). In contrast, ZnPT-treated eggs display condensed chromosomes (h) adjacent to an area of concentrated, cortical F-actin (i). α-tubulin is organized in a metaphase-like configuration (j) around the chromosomes (k, merged image). This layout mirrors the subcellular arrangement in unfertilized eggs (l-o), which also display condensed chromosomes (l) overlaid with an actin cap (m), surrounded by a metaphase spindle (n). Scale bar=80 μm (b, c) or 25 μm (d-o).
FIG. 12 shows perturbation of intracellular zinc availability causes activation of the egg. Following 8 hours of culture, control eggs maintain a metaphase II spindle (a, sp) with individual chromosomes aligned at the metaphase plate (b). In contrast, eggs exposed to 10 μM TPEN for the same period artificially activate as indicated by the formation of an intact pronucleus (c, pn) with decondensed chromatin surrounding a nucleolus (d). The number of eggs displaying a pronucleus is significantly higher in the TPEN-treated group (e).
FIG. 13 shows the chemical structure of an exemplary Zn-responsive probe of the present invention (A, A zinc-chelating ligand containing five potential zinc binding groups; B, BODIPY fluorophore; C, ethanol ether based linker chain; D, amine group that enables attachment of this fluorophore to carboxylate-modified surfaces via amide-coupling chemistries).