Avian eggshell formation reveals a new paradigm for vertebrate mineralization via vesicular amorphous calcium carbonate
2020; Elsevier BV; Volume: 295; Issue: 47 Linguagem: Inglês
10.1074/jbc.ra120.014542
ISSN1083-351X
AutoresLilian Stapane, Nathalie Le Roy, Jacky Ezagal, Alejandro B. Rodríguez‐Navarro, Valérie Labas, Lucie Combes‐Soia, Maxwell T. Hincke, Joël Gautron,
Tópico(s)Turtle Biology and Conservation
ResumoAmorphous calcium carbonate (ACC) is an unstable mineral phase, which is progressively transformed into aragonite or calcite in biomineralization of marine invertebrate shells or avian eggshells, respectively. We have previously proposed a model of vesicular transport to provide stabilized ACC in chicken uterine fluid where eggshell mineralization takes place. Herein, we report further experimental support for this model. We confirmed the presence of extracellular vesicles (EVs) using transmission EM and showed high levels of mRNA of vesicular markers in the oviduct segments where eggshell mineralization occurs. We also demonstrate that EVs contain ACC in uterine fluid using spectroscopic analysis. Moreover, proteomics and immunofluorescence confirmed the presence of major vesicular, mineralization-specific and eggshell matrix proteins in the uterus and in purified EVs. We propose a comprehensive role for EVs in eggshell mineralization, in which annexins transfer calcium into vesicles and carbonic anhydrase 4 catalyzes the formation of bicarbonate ions (HCO3−), for accumulation of ACC in vesicles. We hypothesize that ACC is stabilized by ovalbumin and/or lysozyme or additional vesicle proteins identified in this study. Finally, EDIL3 and MFGE8 are proposed to serve as guidance molecules to target EVs to the mineralization site. We therefore report for the first-time experimental evidence for the components of vesicular transport to supply ACC in a vertebrate model of biomineralization. Amorphous calcium carbonate (ACC) is an unstable mineral phase, which is progressively transformed into aragonite or calcite in biomineralization of marine invertebrate shells or avian eggshells, respectively. We have previously proposed a model of vesicular transport to provide stabilized ACC in chicken uterine fluid where eggshell mineralization takes place. Herein, we report further experimental support for this model. We confirmed the presence of extracellular vesicles (EVs) using transmission EM and showed high levels of mRNA of vesicular markers in the oviduct segments where eggshell mineralization occurs. We also demonstrate that EVs contain ACC in uterine fluid using spectroscopic analysis. Moreover, proteomics and immunofluorescence confirmed the presence of major vesicular, mineralization-specific and eggshell matrix proteins in the uterus and in purified EVs. We propose a comprehensive role for EVs in eggshell mineralization, in which annexins transfer calcium into vesicles and carbonic anhydrase 4 catalyzes the formation of bicarbonate ions (HCO3−), for accumulation of ACC in vesicles. We hypothesize that ACC is stabilized by ovalbumin and/or lysozyme or additional vesicle proteins identified in this study. Finally, EDIL3 and MFGE8 are proposed to serve as guidance molecules to target EVs to the mineralization site. We therefore report for the first-time experimental evidence for the components of vesicular transport to supply ACC in a vertebrate model of biomineralization. Biomineralization is a ubiquitous process by which living organisms produce minerals that they use for many different functions (i.e. protection, gravity sensing) (1Lowenstam H.A. Weiner S. On Biomineralization. Oxford University Press, New York1989Crossref Google Scholar). The formation of calcium carbonate (CaCO3) or phosphate biominerals requires high local concentrations of calcium. Transient amorphous mineral phases that are highly soluble and reactive are a source of ions or a precursor phase for the formation of complex-shaped crystalline biomineral structures. Amorphous calcium carbonate (ACC) is a metastable polymorph of CaCO3, and can provide high concentrations of ions for rapid physiological calcite and aragonite biomineralization (2Ziegler A. 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Among vertebrates, the avian class (Aves) appeared around 91 million years ago and is divided into Paleognathae (ancient birds such as ostrich, rhea, and emu) and Neognathae (modern birds such as chicken, turkey, or zebra finch) (12Claramunt S. Cracraft J. A new time tree reveals Earth history's imprint on the evolution of modern birds.Sci. Adv. 2015; 1: e150100510.1126/sciadv.1501005Crossref PubMed Google Scholar). All birds produce eggs with a hard-mineral shell composed of CaCO3 in the form of calcite, which is critical for development of the embryo within an autonomous chamber (13Hincke M.T. Da Silva M. Guyot N. Gautron J. McKee M.D. Guabiraba-Brito R. Réhault-Godbert S. Dynamics of structural barriers and innate immune components during incubation of the avian egg: critical interplay between autonomous embryonic development and maternal anticipation.J. Innate Immun. 2019; 11 (30391943): 111-12410.1159/000493719Crossref PubMed Scopus (9) Google Scholar). 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This process requires transport of large amounts of calcium and carbonate to the site of eggshell calcification; these ions are continuously supplied from the blood across the uterine epithelium (24Nys Y. Le Roy N. Calcium homeostasis and eggshell biomineralization in female chicken.in: Feldman D. Vitamin D. Academic Press, Cambridge2018: 361-382Crossref Scopus (8) Google Scholar). The active transepithelial transfer of calcium and carbonate is well described and constitutes the current model for eggshell calcification (21Jonchère V. Brionne A. Gautron J. Nys Y. Identification of uterine ion transporters for mineralisation precursors of the avian eggshell.BMC Physiol. 2012; 12 (22943410): 1010.1186/1472-6793-12-10Crossref PubMed Scopus (60) Google Scholar, 25Brionne A. Nys Y. Hennequet-Antier C. Gautron J. Hen uterine gene expression profiling during eggshell formation reveals putative proteins involved in the supply of minerals or in the shell mineralization process.BMC Genomics. 2014; 15 (24649854): 22010.1186/1471-2164-15-220Crossref PubMed Scopus (55) Google Scholar). Alternatively, transport of stabilized ACC mineral in vesicles has been proposed (9Mass T. Giuffre A.J. Sun C.Y. Stifler C.A. Frazier M.J. Neder M. Tamura N. Stan C.V. Marcus M.A. Gilbert P. Amorphous calcium carbonate particles form coral skeletons.Proc. Natl. Acad. Sci. U.S.A. 2017; 114 (28847944): E7670-E767810.1073/pnas.1707890114Crossref PubMed Scopus (0) Google Scholar). In chicken eggshell mineralization, the important role of ACC has been described (26Rodríguez-Navarro A.B. Marie P. Nys Y. Hincke M.T. Gautron J. Amorphous calcium carbonate controls avian eggshell mineralization: a new paradigm for understanding rapid eggshell calcification.J. Struct. Biol. 2015; 190 (25934395): 291-30310.1016/j.jsb.2015.04.014Crossref PubMed Scopus (67) Google Scholar). During the earliest stage, massive deposits of ACC accumulate at specific nucleation sites (mammillary knobs) on the eggshell membrane. Subsequently, ACC transforms into calcite crystals. Moreover, progressive ACC dissolution continuously supplies local ions to support the rapid growth of columnar calcite crystals that constitute the palisade layer (26Rodríguez-Navarro A.B. Marie P. Nys Y. Hincke M.T. Gautron J. Amorphous calcium carbonate controls avian eggshell mineralization: a new paradigm for understanding rapid eggshell calcification.J. Struct. Biol. 2015; 190 (25934395): 291-30310.1016/j.jsb.2015.04.014Crossref PubMed Scopus (67) Google Scholar). In a recent study, we used bioinformatics tools, mRNA levels, and protein quantification to explore the role of EDIL3 and MFGE8 in chicken eggshell biomineralization. We hypothesized that EDIL3 and MFGE8 bind to EVs budding from uterine cells into the uterine fluid, to guide vesicular transport of stabilized ACC for delivery to the mineralizing site and moreover prevent nonspecific precipitation (27Stapane L. Le Roy N. Hincke M.T. Gautron J. The glycoproteins EDIL3 and MFGE8 regulate vesicle-mediated eggshell calcification in a new model for avian biomineralization.J. Biol. Chem. 2019; 294 (31358619): 14526-1454510.1074/jbc.RA119.009799Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). To test this hypothesis, in the current study we have used transmission EM (TEM) to investigate exocytosis activity at the apical plasma membrane of uterine cells that could be a source of EVs in the uterine fluid. We quantified mRNA levels of a variety of validated EV components in the oviduct segments and other tissues. We purified EVs from UF and demonstrated the presence of key vesicular proteins in these vesicles. Finally, spectroscopic techniques (energy-dispersive X-ray spectroscopy (EDX) and electron energy loss spectroscopy (EELS)) probed for ACC inside these EVs. This experimental study is the first to demonstrate vesicular transport of ACC in vertebrates, which we propose supports the rapid eggshell biomineralization process in birds. The presence of vesicles in the tissues and milieu involved in shell mineralization was investigated by TEM. Ultra-thin negatively stained sections of uterus epithelium were examined by TEM to investigate exocytosis activity adjacent to the luminal site of mineralization (Fig. 1). Uterine-ciliated cells possess numerous vacuolar and vesicular structures as well as dense and light granules (Fig. 1A). At higher magnification (Fig. 1, B–D) we observed vesicles in the cell cytoplasm, their accumulation at the apical plasma membrane, and their budding to generate EVs in the adjacent luminal uterine fluid (Fig. 1, C and D). Fig. 1, E and F, show EVs in the uterine fluid, with vesicle diameters in the 100-400 nm range. Vesicle membranes (rich in glycoproteins, proteins, and lipids) are stained by uranyl acetate and appear bright (high electron-density) versus their interiors that are darker (low electron density). However, some internal regions of vesicles also appeared electron-dense due to mineral deposits (see below). These TEM observations demonstrated the presence of vesicles in the luminal uterine fluid adjacent to the apical region of uterine cells. The uterine epithelium contains ciliated and nonciliated cells, and the same results were observed in proximity to nonciliated cells (data not shown). UF was also examined by TEM (Figure 2, Figure 3), where numerous EVs varying in diameter from 100 to 500 nm were observed (Figs. 2, A and C, and 3A).Figure 3TEM on uterine fluid, EDS and SAED analyses. A, TEM of EVs observed in UF fraction at 16 h p.o. with associated mapping of (B) oxygen element, (C) calcium element, and (D) carbon element; as detected by the EDS analysis. E, EDS spectrum of the EVs observed in UF and shown in A. Filled white arrowhead indicates the calcium peak (3.690 keV) of EV, whereas striped and empty white arrowheads show carbon (0.277 keV) and oxygen (0.525 keV) peaks, respectively. Boxes point out the areas of EDS analysis. F and G, SAED pattern of the mineral detected in EV of UF. Diffuse scattering rings are characteristic of the ACC mineral. Bars: A to D = 500 nm, F = 1 μm, G = 51 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2TEM and EELS on UF. A and C, TEM micrograph of EVs observed in UF. Black arrowheads indicate the EVs. White boxes indicate EELS analysis area of B and D graphs. B and D, EELS spectroscopy on EVs from UF. The carbon K-edge shows three major peaks at 290.3, 295.5, and 301.5 electron volts (eV), characteristic of CaCO3. The peak at 285.0 from the carbon K-edge reflects the presence of organic material. The two peaks 349.3 and 352.6 eV define the calcium L2,3-edge of EELS. The oxygen K-edge displays a major peak at 540.0 specific to the carbonate group (CO32−) and two other peaks at 534.0 and 545.5 indicate C = O bond. E, TEM micrograph of EV, observed in UF with associated mapping of calcium element (F) (324-355 eV). G, organic carbon (280-290 eV) and H, calcium + carbon combined elements detected by the EELS analysis. Bars: A = 1 μm, C = 200 nm, E to H = 500 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) At collection, uterine fluid was immediately frozen in liquid nitrogen to preserve EVs and their cargo. Uterine fluid vesicles containing electron dense mineral deposits were observed by TEM and analyzed by EELS (Fig. 2) and EDS (Fig. 3). EELS spectra show characteristics peaks from the calcium L2,3-edge (349.3 and 352.6 eV), carbon K-edge (285.0, 290.3, 295.5, and 301.5 eV), and oxygen K-edge (540.0 eV) (Fig. 2, B and D, Table S1). All carbon K-edge and oxygen K-edge peaks, except 285.0 eV, are characteristics of carbonate groups (28DeVol R.T. Metzler R.A. Kabalah-Amitai L. Pokroy B. Politi Y. Gal A. Addadi L. Weiner S. Fernandez-Martinez A. Demichelis R. Gale J.D. Ihli J. Meldrum F.C. Blonsky A.Z. Killian C.E. et al.Oxygen spectroscopy and polarization-dependent imaging contrast (PIC)-mapping of calcium carbonate minerals and biominerals.J. Phys. Chem. B. 2014; 118 (24821199): 8449-845710.1021/jp503700gCrossref PubMed Scopus (31) Google Scholar, 29Garvie L.A.J. Craven A.J. Brydson R. Use of electron-energy-loss near-edge fine-structure in the study of minerals.Am. Mineralogist. 1995; 80: 1132-142510.2138/am-1995-11-1204Crossref Scopus (69) Google Scholar). Thus, this analysis confirmed that the mineral deposits are calcium carbonate. Moreover, the 285.0 eV peak from the carbon K-edge is characteristic of amorphous carbon (from organics) and of C = C bonds, whereas the two peaks from the oxygen K-edge (534.0 and 545.5 eV) are also specific to C = O bonds (30Macías-Sánchez E. Willinger M.G. Pina C.M. Checa A.G. Transformation of ACC into aragonite and the origin of the nanogranular structure of nacre.Sci. Rep. 2017; 710.1038/s41598-017-12673-0Crossref PubMed Scopus (14) Google Scholar). Therefore, EELS spectra confirmed the presence in the uterine EVs of an organic phase (phospholipids and proteins) as well as calcium carbonate mineral deposits, although low spectral resolution (0.25 eV) of the EELS detector did not allow differentiating among different polymorphs. However, selected area electron diffraction (SAED) of the vesicle mineral deposits showed diffuse rings indicative of the amorphous nature of calcium carbonate mineral (Fig. 3, F and G). To determine the distribution of the organics and CaCO3 mineral deposits, the carbon peak from the organics (237-290 eV) and calcium peaks (324-355 eV) were selected for mapping (Fig. 2, E–H). The carbon and calcium maps showed that the organic phase (C = C) was concentrated at the vesicle periphery (Fig. 2G), whereas calcium was concentrated within the vesicles. Merger of the carbon and calcium maps (Fig. 2H) clearly shows that the EV organic membranes (phospholipid and proteins) enclose the amorphous calcium carbonate mineral deposits. The distribution of calcium, carbon, and oxygen in uterine fluid vesicles were also analyzed using EDS (Fig. 3). Oxygen, carbon, and calcium elements co-localized within EVs (Fig. 3, A–D). A high C background signal is notable, as samples were mounted on carbon-coated TEM grids. The difference between EDS spectrum 1 (EV), spectrum 2 (EV), and spectrum 3 (background) confirmed the presence of significant amounts of calcium and oxygen inside the EVs, compared with the background signal (Fig. 3E). Indeed, calcium and oxygen signals in the EV were 3- to 5-times higher (spectrum 1) than the background (spectrum 3). We evaluated the literature on bone and cartilage extracellular vesicles (31Balcerzak M. Malinowska A. Thouverey C. Sekrecka A. Dadlez M. Buchet R. Pikula S. Proteome analysis of matrix vesicles isolated from femurs of chicken embryo.Proteomics. 2008; 8 (18095356): 192-20510.1002/pmic.200700612Crossref PubMed Scopus (60) Google Scholar, 32Shapiro I.M. Landis W.J. Risbud M.V. Matrix vesicles: are they anchored exosomes?.Bone. 2015; 79 (25980744): 29-3610.1016/j.bone.2015.05.013Crossref PubMed Google Scholar, 33Rosenthal A.K. Gohr C.M. Ninomiya J. Wakim B.T. 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A total of 33 genes coding for proteins involved in vesicular transport were selected, corresponding to proteins involved as calcium channels to supply calcium, bicarbonate supplier/transporter, chaperone molecules, addressing molecules, intracellular trafficking proteins, extracellular biogenesis and release, and signaling proteins (Table S2). mRNA levels for these 33 genes were quantified in different tissues and organs, namely oviduct segments, bone, duodenum, kidney, and liver (Table 1, Fig. 4). Tissue samples from four specialized oviduct regions were collected to evaluate the mRNA level of EV markers associated with egg white deposition (Ma), eggshell membrane formation (WI), and shell calcification (RI, Ut). Bone was selected as a mineralized tissue (with hydroxyapatite) where EVs have been demonstrated (31Balcerzak M. Malinowska A. Thouverey C. Sekrecka A. Dadlez M. Buchet R. Pikula S. 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Duodenum (D) and kidney (K) exhibit active ion transport activity without any associated calcification. Finally, liver (L) was selected as an important organ involved in general metabolism. Comparisons of quantified mRNA levels in these organs and tissues were displayed using a heat map diagram (Fig. 4). Z-scores are expressed in terms of mean ± S.D. for each gene. Consequently, the color scale indicates the relative variation of each gene in the different tissues. We observed the highest Z-scores in oviduct segments (Ma, WI, RI, and Ut), for 20 vesicular genes (Anxa1, Anxa2, Anxa8, Ap1g1, Cd9, Cd82, Edil3, Hspa8, Itgb1, Pdcd6ip, Rab5a, Rab27a, Sdcbp, Tsg101, Vamp3, Vamp7, Vps4, Vps26a, Ywhah, and Ywhaz) compared with the other tissues (Clusters 6 to 8, Fig. 4). The mRNA level of genes was also analyzed using ANOVA and Tukey pairwise analysis (Table 1). With the notable exception of Ap1g1, all other genes with highest Z-scores in uterus were also significantly different in the same uterine tissue using ANOVA and pairwise analysis (Table 1). Additionally, these statistical tests show that Itgb1, Sdcbp, Vamp7, and Vps4b were also significantly overexpressed in one or several other tissues (bone (B), kidney (K), and duodenum (D)). Anxa5, Anxa11, Ap1g1, Hsp90b, and Rab7a were not differentially expressed in the various tissues tested. Both Rab11a and Slc4a7 were significantly over-expressed in D, whereas Ca2 and Anxa7 were significantly overexpressed in B and K, respectively, to other tissues. Arf6 exhibited a significantly higher mRNA level in D and uterus. The remaining three genes Anxa6, Ralb, and Vcp did not exhibit significantly different mRNA levels (Table 1).Table 1Normalized vesicular mRNA levels in the four oviduct regions and other tissuesFunctionTissuesOviduct segmentsGeneBDKLMaWIRIUtANOVA p valueCalcium channelAnxa10.205 ± 0.200cd0.002 ± 0.001d0.009 ± 0.004d0.001 ± 0.001d0.194 ± 0.066 cd2.190 ± 0.749b3.338 ± 0.768a0.891 ± 0.320c<0.001Anxa20.307 ± 0.240cd0.521 ± 0.180 cd0.006 ± 0.009d0.001 ± 0.002d0.543 ± 0.340 cd1.208 ± 0.805bc2.884 ± 1.247a1.904 ± 0.459ab<0.001Anxa50.431 ± 0.3590.401 ± 0.2890.189 ± 0.1731.462 ± 0.8282.001 ± 2.9200.560 ± 0.2981.405 ± 1.0440.298 ± 0.1930.068Anxa62.116 ± 1.522ab0.446 ± 0.282b0.313 ± 0.163b1.473 ± 0.558ab1.328 ± 1.140ab0.478 ± 0.375ab0.347 ± 0.147b0.543 ± 0.437ab0.008Anxa70.206 ± 0.081cd1.436 ± 0.306b7.074 ± 1.170a0.191 ± 0.059d0.623 ± 0.330bcd0.676 ± 0.410bcd1.122 ± 0.284bc1.450 ± 0.526b<0.001Anxa80.026 ± 0.029d0.001 ± 0.001d0.001 ± 0.001d0.001 ± 0.001d1.669 ± 0.922bc2.513 ± 1.205ab3.958 ± 1.720a0.979 ± 0.344 cd<0.001Anxa110.214 ± 0.0440.562 ± 0.1520.644 ± 0.2930.9
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