JBC Papers in Press. Published on August 18, 2020 as Manuscript RA120.014542

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JBC Papers in Press. Published on August 18, 2020 as Manuscript RA120.014542
JBC Papers in Press. Published on August 18, 2020 as Manuscript RA120.014542
     The latest version is at https://www.jbc.org/cgi/doi/10.1074/jbc.RA120.014542

  Avian eggshell formation reveals a new paradigm for vertebrate mineralization via vesicular
                                amorphous calcium carbonate.
  Lilian Stapane1, Nathalie Le Roy1, Jacky Ezagal1, Alejandro B. Rodriguez-Navarro2, Valérie
               Labas3, Lucie Combes-Soia3, Maxwell T. Hincke4, Joël Gautron1*

From 1BOA, INRAE, Université de Tours, 37380 Nouzilly, France ; 2Departmento de Mineralogia y
Petrologia, Universidad de Granada, 18071 Granada, Spain ; 3UMR PRC, INRAE 85, CNRS 7247,
Université de Tours, IFCE, 37380 Nouzilly, France ; 4Department of Innovation in Medical Education,
and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada K1H8M5

                    Running title: EV-packaged ACC for avian eggshell formation

* To whom correspondence should be addressed: BOA, INRAE, Université de Tours, 37380 Nouzilly,
France; joel.gautron@inrae.fr; Tel.+33 2 47 42 75 40.

Keywords: Biomineralization, Avian eggshell, extracellular vesicle, ACC transport, matrix proteins.

                                                       transport to supply ACC in a vertebrate model

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ABSTRACT                                               of biomineralization.
         Amorphous calcium carbonate (ACC)
is an unstable mineral phase, which is
progressively transformed into aragonite or                     Biomineralization is a ubiquitous
calcite in biomineralization of marine                 process by which living organisms produce
invertebrate shells or avian eggshells,                minerals that they use for many different
respectively. We have previously proposed a            functions (i.e., protection, gravity sensing) (1).
model of vesicular transport to provide                The formation of calcium carbonate (CaCO3) or
stabilized ACC in chicken uterine fluid where          phosphate biominerals requires high local
eggshell mineralization takes place. Herein, we        concentrations       of     calcium.    Transient
report further experimental support for this           amorphous mineral phases that are highly
model. We confirmed the presence of                    soluble and reactive are a source of ions or a
extracellular vesicles (EVs) using transmission        precursor phase for the formation of complex
electron microscopy and showed high levels of          shaped crystalline biomineral structures.
mRNA of vesicular markers in the oviduct               Amorphous calcium carbonate (ACC) is a
segments where eggshell mineralization occurs.         metastable polymorph of CaCO3, and can
We also demonstrate that EVs contain ACC in            provide high concentrations of ions for rapid
uterine fluid using spectroscopic analysis.            physiological       calcite      and    aragonite
Moreover,              proteomics           and        biomineralization (2). The high solubility of
immunofluorescence confirmed the presence of           these metastable phases requires stabilization
major vesicular, mineralization-specific and           by regulatory molecules (3-9). Extracellular
eggshell matrix proteins in the uterus and in          vesicles (EVs) are candidates to play a role in
purified EVs. We propose a comprehensive role          the stabilization and delivery of ACC (6-9).
for EVs in eggshell mineralization, in which           However, experimental evidence for EVs in
annexins transfer calcium into vesicles and            ACC-mediated calcification in CaCO3
carbonic anhydrase 4 catalyzes the formation of        biominerals is only available for invertebrate
bicarbonate ions (HCO3-), for accumulation of          structures such as sea urchin spines (calcite) and
ACC in vesicles. We hypothesize that ACC is            molluscan shells (4,10,11).
stabilized by ovalbumin and/or lysozyme or                      Amongst vertebrates, the avian class
additional vesicle proteins identified in this         (Aves) appeared around 91 million years ago
study. Finally, EDIL3 and MFGE8 are                    and is divided into Paleognathae (ancient birds
proposed to serve as guidance molecules to             such as ostrich, rhea and emu) and Neognathae
target EVs to the mineralization site. We              (modern birds such as chicken, turkey or zebra
therefore report for the first-time experimental       finch) (12). All birds produce eggs with a hard-
evidence for the components of vesicular               mineral shell composed of CaCO3in the form of
                                                       calcite, that is critical for development of the

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JBC Papers in Press. Published on August 18, 2020 as Manuscript RA120.014542
New model for avian eggshell formation via stabilized ACC

embryo within an autonomous chamber (13). In             of EVs in the uterine fluid. We quantified
addition to defense against physical aggression,         mRNA levels of a variety of validated EV
the eggshell protects the egg contents against           components in the oviduct segments and other
microbial contamination, regulates water and             tissues. We purified EVs from UF and
gaseous exchange, and is a calcium source for            demonstrated the presence of key vesicular
embryonic bone calcification (14,15). The                proteins in these vesicles. Finally, spectroscopic
chicken eggshell is a widely utilized                    techniques (EDX and EELS) probed for ACC
experimental model for biomineralization.                inside these EVs. This experimental study is the
Eggshell formation occurs in the distal part of          first to demonstrate vesicular transport of ACC
the hen oviduct (red isthmus and uterus) and is          in vertebrates, which we propose supports the
one of the fastest known processes of vertebrate         rapid eggshell biomineralization process in
biomineralization (16-18). In chickens, 6 g of           birds.
CaCO3 are rapidly deposited in a very short time
(< 18 h; deposition rate of 0.32 g/h) (19). During       Results
this extracellular process, the uterine cells            Transmission electron microscopy of uterine
secrete organic and mineral eggshell precursors          epithelial cells and uterine fluid
into the uterine fluid (UF) where mineralization                  The presence of vesicles in the tissues
takes place (20-22). Both mineral and organic            and milieu involved in shell mineralization was
precursors interact to produce the specific              investigated      by    transmission     electron
                                                         microscopy (TEM).          Ultra-thin negatively

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eggshell texture and its resulting mechanical
properties (17,23). This process requires                stained sections of uterus epithelium were
transport of large amounts of calcium and                examined by TEM to investigate exocytosis
carbonate to the site of eggshell calcification;         activity adjacent to the luminal site of
these ions are continuously supplied from the            mineralization (Fig.1). Uterine ciliated cells
blood across the uterine epithelium (24). The            possess numerous vacuolar and vesicular
active transepithelial transfer of calcium and           structures as well as dense and light granules
carbonate is well described and constitutes the          (Fig.1A). At higher magnification (Fig.1B-D)
current model for eggshell calcification (21,25).        we observed vesicles in the cell cytoplasm, their
Alternatively, transport of stabilized ACC               accumulation at the apical plasma membrane,
mineral in vesicles has been proposed (9).               and their budding to generate EVs in the
         In chicken eggshell mineralization, the         adjacent luminal uterine fluid (Fig.1C-D). Fig.
important role of ACC has been described (26).           1E, F show EVs in the uterine fluid, with vesicle
During the earliest stage, massive deposits of           diameters in the 100-400 nm range. Vesicle
ACC accumulate at specific nucleation sites              membranes (rich in glycoproteins, proteins,
(mammillary knobs) on the eggshell membrane.             lipids) are stained by uranyl acetate and appear
Subsequently, ACC transforms into calcite                bright (high electron-density) versus their
crystals.    Moreover,       progressive     ACC         interiors that are darker (low electron density).
dissolution continuously supplies local ions to          However, some internal regions of vesicles also
support the rapid growth of columnar calcite             appeared electron-dense due to mineral deposits
crystals that constitute the palisade layer (26).        (see section below). These TEM observations
In a recent study, we used bioinformatics tools,         demonstrated the presence of vesicles in the
mRNA levels and protein quantification to                luminal uterine fluid adjacent to the apical
explore the role of EDIL3 and MFGE8 in                   region of uterine cells. The uterine epithelium
chicken eggshell biomineralization. We                   contains ciliated and non-ciliated cells, and the
hypothesized that EDIL3 and MFGE8 bind to                same results were observed in proximity to non-
EVs budding from uterine cells into the uterine          ciliated cells (data not shown). UF was also
fluid, in order to guide vesicular transport of          examined by TEM (Fig.2 and 3), where
stabilized ACC for delivery to the mineralizing          numerous EVs varying in diameter from 100 to
site and moreover prevent non-specific                   500 nm were observed (Fig.2A, C and Fig.3A).
precipitation (27).                                      TEM and spectroscopic analysis of
         In order to test this hypothesis, in the        extracellular vesicle mineral deposits
current study we have used transmission                          At collection, uterine fluid was
electron microscopy (TEM) to investigate                 immediately frozen in liquid nitrogen in order
exocytosis activity at the apical plasma                 to preserve EVs and their cargo. Uterine fluid
membrane of uterine cells that could be a source         vesicles containing electron dense mineral

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JBC Papers in Press. Published on August 18, 2020 as Manuscript RA120.014542
New model for avian eggshell formation via stabilized ACC

deposits were observed by TEM and analyzed            signals in the EV were 3- to 5-times higher
by electron energy loss spectroscopy (EELS,           (spectrum 1), than the background (spectrum 3).
Fig.2),     and     energy-dispersive     X-ray
spectroscopy (EDS, Fig. 3).                           Vesicular mRNA levels in various Gallus
        EELS spectra show characteristics             gallus tissues
peaks from the calcium L2,3-edge (349.3 and                    We evaluated the literature on bone and
352.6 eV), carbon K-edge (285.0, 290.3, 295.5         cartilage extracellular vesicles (31-34), EVs
and 301.5 eV) and oxygen K-edge (540.0 eV)            (35-38), and the Vesiclepedia database
(Fig.2B, D, Table S1). All carbon K-edge and          (www.microvesicles.org/), which lists the top
oxygen K-edge peaks, except 285.0 eV, are             100 proteins that are identified in EVs (39). A
characteristics of carbonate groups (28,29).          total of 33 genes coding for proteins involved in
Thus, this analysis confirmed that the mineral        vesicular       transport      were      selected,
deposits are calcium carbonate. Moreover, the         corresponding to proteins involved as calcium
285.0 eV peak from the carbon K-edge is               channels to supply calcium, bicarbonate
characteristic of amorphous carbon (from              supplier/transporter, chaperone molecules,
organics) and of C=C bonds whereas the two            addressing molecules, intracellular trafficking
peaks from the oxygen K-edge (534.0 eV and            proteins, extracellular biogenesis and release,
545.5 eV) are also specific to C=O bonds (30).        and signaling proteins (Table S2). Messenger
Therefore, EELS spectra confirmed the                 RNA levels for these 33 genes were quantified
                                                      in different tissues and organs, namely oviduct

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presence in the uterine EVs of an organic phase
(phospholipids and proteins) as well as calcium       segments, bone, duodenum, kidney and liver
carbonate mineral deposits, although low              (Table 1, Fig.4). Tissue samples from four
spectral resolution (0.25 eV) of the EELS             specialized oviduct regions were collected to
detector did not allow differentiating among          evaluate mRNA level of EV markers associated
different polymorphs. However, selected area          with egg white deposition (Ma), eggshell
electron diffraction (SAED) of the vesicle            membrane formation (WI), and shell
mineral deposits showed diffuse rings                 calcification (RI, Ut). Bone was selected as a
indicative of the amorphous nature of calcium         mineralized tissue (with hydroxyapatite) where
carbonate mineral (Fig. 3F, G).                       EVs have been demonstrated (31,32,40,41).
        To determine the distribution of the          Duodenum (D) and kidney (K) exhibit active
organics and CaCO3 mineral deposits, the              ion transport activity without any associated
carbon peak from the organics (237-290 eV)            calcification. Finally, liver (L) was selected as
and calcium peaks (324-355 eV) were selected          an important organ involved in general
for mapping (Fig. 2E-H). The carbon and               metabolism. Comparisons of quantified mRNA
calcium maps showed that the organic phase            levels in these organs and tissues were
(C=C) was concentrated at the vesicle periphery       displayed using a heatmap diagram (Fig.4). Z-
(Fig. 2G) whereas calcium was concentrated            scores are expressed in terms of standard
within the vesicles. Merger of the carbon and         deviations from their means for each gene.
calcium maps (Fig. 2H) clearly shows that the         Consequently, the color scale indicates the
EV organic membranes (phospholipid and                relative variation of each gene in the different
proteins) enclose the amorphous calcium               tissues. We observed the highest Z-scores in
carbonate mineral deposits.                           oviduct segments (Ma, WI, RI, Ut), for 20
        The distribution of calcium, carbon and       vesicular genes (Anxa1, Anxa2, Anxa8,Ap1g1,
oxygen in uterine fluid vesicles were also            Cd9, Cd82, Edil3, Hspa8, Itgb1, Pdcd6ip,
analyzed using energy-dispersive X-ray                Rab5a, Rab27a, Sdcbp, Tsg101, Vamp3,
spectroscopic (EDS) (Fig. 3). Oxygen, carbon          Vamp7, Vps4, Vps26a, Ywhah and Ywhaz)
and calcium elements co-localized within EVs          compared to the other tissues (Clusters 6 to 8,
(Fig. 3A-D). A high C background signal is            Fig.4). Messenger RNA level of genes was also
notable, as samples were mounted on carbon            analyzed using ANOVA and Tukey pairwise
coated TEM grids. The difference between EDS          analysis (Table 1). With the notable exception
spectrum 1 (EV), spectrum 2 (EV) and spectrum         of Ap1g1, all other genes with highest Z-scores
3 (background) confirmed the presence of              in uterus were also significantly different in the
significant amounts of calcium and oxygen             same uterine tissue using ANOVA and pairwise
inside the EVs, compared to the background            analysis (Table 1). Additionally, these statistical
signal (Fig. 3E). Indeed, calcium and oxygen          tests show that Itgb1, Sdcbp, Vamp7 and Vps4b

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JBC Papers in Press. Published on August 18, 2020 as Manuscript RA120.014542
New model for avian eggshell formation via stabilized ACC

were also significantly over-expressed in one or        fraction and SEP (Fig. 5A). MFGE8 exhibited a
several other tissues (bone (B), kidney (K), and        single band around 75-kDa (53-kDa predicted
duodenum (D)). Anxa5, Anxa11, Ap1g1,                    molecular weight) in all samples (Fig.5B). The
Hsp90b and Rab7a were not differentially                higher than predicted molecular mass observed
expressed in the various tissues tested. Both           for both EDIL3 and MFGE8 could be due to
Rab11a and Slc4a7 were significantly over-              post-translational modification such as
expressed in D, while Ca2 and Anxa7 were                glycosylation (27). An MFGE8 immunoband
significantly over-expressed in B and K                 was detected in uterine fluid lacking vesicles,
respectively to other tissues. Arf6 exhibited a         indicating that MFGE8 is soluble in uterine
significant higher mRNA level in D and uterus.          fluid, unlike EDIL3.
The remaining three genes Anxa6, Ralb, and
Vcp did not exhibit significantly different             Immunofluorescence in uterus for key
mRNA levels (Table 1).                                  proteins proposed for UF vesicular transport
                                                                 Immunofluorescence analysis was
Proteomic and Western blot analyses of EVs              performed on chicken uterus sections to localize
         To determine the protein composition           six proteins (ANXA1, ANXA2, ANXA8, CA4,
of EVs isolated from the UF, we carried out             EDIL3, PDCD6IP) proposed to play key roles
proteomics analysis using nanoLC-MS/MS and              in vesicular transport within the tissue
verified these observations with Western                responsible for eggshell mineralization (Fig.6).
blotting techniques. Twenty-nine non-                   Analyses were performed at five defined time

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redundant proteins were identified in our               points in the eggshell mineralization process.
proteomics analysis (Table 2). Amongst them,            Time points at 5 and 6 h p.o. correspond to the
seven proteins correspond to predicted major            massive accumulation of ACC at the earliest
actors in the hypothetical vesicular transport of       stages of shell formation. At 7 h p.o., ACC is
ACC (Carbonic Anhydrase-4 (CA4), EGF-like               progressively transformed in calcite aggregates
repeat and discoidin I-like domain-containing           whereas calcite crystals with preferential
protein 3 (EDIL3), ezrin (EZR), programmed              orientation are fusing at 10 h p.o. to form the
cell death 6-interacting protein (PDCD6IP),             compact palisade layer strongly deposited at 16
syntenin-1, ovalbumin (OVA) and lysozyme C              h p.o.). Negative controls demonstrated the
(LYZ); Tables 2 and 3) (27). In addition, several       absence of nonspecific signals in uterus for all
immunoglobulins and other proteins already              antibodies. EDIL3 and ANXA1 protein levels
identified in chicken eggshell matrix                   were maximum at 5 h and 6 h p.o., when
(ovotransferrin,      ovalbumin-related        X,       massive ACC is deposited and then decreased
ovalbumin-related Y, ovocalyxin-25, clusterin,          from 7 h to 16 h p.o. (Fig.6). EDIL3 is present
ovomucin, vitelline membrane outer layer                in the tubular glands of the lamina propria,
protein 1, ovostatin, ovoglobulinG2, prominin-          whereas ANXA1 was localized to the
1-A, alpha-2-macroglobulin, aminopeptidase              epithelium. A faint immunostaining was
N) were detected (Table 2). Finally, the                obtained for ANXA2 at 5 h p.o., while the signal
angiotensin-converting enzyme precursor                 was stronger at 6 h, 7 h and 10 h p.o. in the
(ACE) was also identified.                              epithelium (Fig.6), then decreased at 16h p.o..
         To    confirm     EDIL3       proteomic        ANXA8 was detected in both tubular glands
identification, we performed Western blot               and epithelium regions at each stage of
analysis on various samples: unfractioned UF            mineralization, with a maximum signal at 10 h
(complete), EV fraction of UF (EV fraction),            p.o. (Fig.6). A strong CA4 signal was observed
depleted UF (ØEV) and soluble eggshell                  in the epithelial cells at 5 h, 6 h when massive
proteins (SEP) (Fig.5A, B). We also carried out         accumulation of ACC occurred and at 7 h p.o.
immunoblotting for milk fat globule-EGF factor          when a dissolution process allowed ACC
8 (MFGE8), since it is another protein                  transformation in calcite, while the signal was
potentially involved in guiding EVs during shell        low at 16 h p.o. PDCD6IP immunofluorescence
mineralization (27). Immunoreactive bands in            revealed that the protein was largely localized
the vesicle fractions were observed using both          in     the     epithelium       throughout   the
antibodies (anti-EDIL3 and anti-MFGE8).                 biomineralization process (5 h to 16 h p.o.)
Using anti-EDIL3 antibodies, a unique immune            (Fig.6).
band was observed around 60-kDa (54-kDa
predicted molecular weight), only in the EV

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JBC Papers in Press. Published on August 18, 2020 as Manuscript RA120.014542
New model for avian eggshell formation via stabilized ACC

Discussion                                              Eggshell mineralization and vesicular
                                                        transport
Vesicles in hen oviduct                                          Five to six g of calcium carbonate are
         EVs are defined as phospholipid-               deposited within an 18 hours period during
enclosed nanoparticles (30–2000 nm) that carry          chicken egg formation. This requires rapid
a complex and variable cargo of biological              delivery of large amounts of calcium and
contents including proteins and nucleic acids           carbonate, representing 10 % (2 g) of hen total
(36). EVs have been categorized as exosomes             body calcium that is exported each day (24).
(30-150 nm) released by exocytosis, apoptotic           Neither component (Ca2+ or HCO3-) is stored in
bodies (50-5000 nm) released at the later stages        the uterus, but rather are continuously supplied
of cell apoptosis and shedding microvesicles or         during eggshell formation from the blood
ectosomes (100-1000 nm) secreted by outward             plasma via transport across the uterine glandular
budding of the plasma membrane (42). Diverse            cells. Mechanisms of transepithelial transfer of
biological functions have been ascribed to EVs,         calcium and bicarbonates have been widely
which vary according to the parental cell from          studied and represent the current accepted
which they were derived and the local                   model for eggshell mineralization (21,24,25).
microenvironment (36). Indeed, EVs have been            Recently, intestinal paracellular transfer of
implicated in numerous process such as tissue           calcium during eggshell formation was
formation as well as in pathological conditions,        demonstrated (52), raising the possibility of

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including vascular calcification and many forms         paracellular transfer of calcium in uterus as
of cancer (36). They have been isolated from            well; however, no experimental data addressing
many biological fluids and recent reviews               this possibility is yet available. The vesicular
highlight their biological significance for             transport of calcium as stabilized ACC is a third
intercellular communication and during                  complementary pathway to transfer calcium and
pathological (43-48).                                   carbonate to the calcification site.
         Recently, EVs have been reported to                     Shell calcification, which takes place in
play potential roles in preserving sperm                a saturated milieu (uterine fluid) (53), is
function within the hen genital tract and mediate       initiated with the accumulation of ACC deposits
interaction with sperm (49). Avian species have         on the eggshell membranes during 5 h – 7 h p.o.
sperm reservoirs called sperm storage tubules           (26). We have recently proposed a novel model
(SST), which are mainly located in the mucosa           for avian eggshell biomineralization involving
of the utero-vaginal junction (UVJ). Exosomes           EVs budding from uterine epithelial cells, that
were observed both at the surface epithelium            transit through the uterine fluid (UF) and deliver
and inside SST using electron microscopy.               stabilized ACC mineral to the eggshell
Moreover, exosomes have been isolated from              membranes and mineralization front (29). One
the avian uterine fluid and then observed using         attractive advantage of this mechanism is that
electron microscopy (49).                               non-specific precipitation in the fluid cannot
         The role of vesicles in the transport of       occur. In this study, we report additional
mineralizing ions during biomineralization has          experimental support for this model. We
been reported in both invertebrate and                  demonstrate the presence of EVs containing
vertebrate systems. The most documented                 ACC in UF, and we define some of the
vesicular mediated mineralization systems are           molecular actors involved in this process
in vertebrate skeleton and teeth, for which             (Fig.7). Moreover, we observed vesicles from
vesicles associated with calcium phosphate              100 to 500 nm in the uterine cell cytoplasm and
have been described (40,41,50). However,                their accumulation at the apical membranes. We
vesicular    mediated      calcium      carbonate       also observed budding from uterine cells to
mineralization has only been reported in                secrete these vesicles into uterine fluid. The size
invertebrates. Sea urchin spicules are the best         of vesicles observed in both fluid and uterine
documented example of invertebrate calcite              cells, and their outward budding, confirmed that
biomineralization mediated by vesicles (6,51).          EVs similar to shedding microvesicles or
The involvement of vesicles was also proposed           ectosomes are present. Secretory activity of
but not confirmed for molluscan shell formation         multivesicular bodies has been described at the
(7) and coral exoskeletons(8).                          hen utero-vaginal junction, where spermatozoa
                                                        are preserved in specialized glands (54).
                                                        Therefore, the distinct oviduct segments seem

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New model for avian eggshell formation via stabilized ACC

to use vesicles for different functions (i.e. sperm        ACC formation. Three of them (Anxa1, -2 and -
preservation and secretion of mineral                      8), were highly expressed in the oviduct
precursors). Moreover, it is intriguing that 250-          segments where eggshell calcification takes
300 nm granules have been detected within the              place (Ut and RI) (Fig. 8), and were
calcium reserve body (CRB) sac of the                      overabundant at the initial phase of shell
mineralized mammillary cones (55), which                   mineralization (5 to 10 h p.o.), compared to later
could correspond to trapped mineralization-                stages. ANXA1 (21,70), ANXA2 (21,25,71-73)
specific EVs.                                              and ANXA8 (70,73) have been reported in
         This study also gives new important               chicken eggshell proteomic and transcriptomic
insight on the stabilization and delivery of ACC           surveys (70). ANXA1, ANXA2 and ANXA8
to the mineralization site where the final                 could bind to EV membranes via their
structure of eggshell is completed. The avian              phospholipid-binding domain (74). These three
eggshell is a highly ordered structure, consisting         annexins constitute, therefore, excellent
of a porous bioceramic resulting from the                  candidates as participants in vesicular transport,
sequential deposition of calcium carbonate and             where they could mediate the entry and
organic matrix in the red isthmus/uterus regions           accumulation of calcium.
of the distal oviduct (15,17,23,56-58). Eggshell                    Bicarbonate is a precursor for the
mineralization results from the aggregation of             calcium carbonate of avian eggshells, and
ACC particles and their transformation into                consequently we investigated carbonic

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calcite crystals to allow a very rapid and                 anhydrase 2 and 4 (CA2 and CA4) and sodium
controlled process. Additionally, ACC, which is            bicarbonate cotransporter 3 (SLC4A7), as
highly unstable under physiological conditions,            bicarbonate suppliers in vesicular transport,
must be stabilised in this milieu. In the present          since these have previously detected in uterine
study, both EELS and EDS spectroscopy                      transcriptomes during eggshell calcification
confirmed the presence of calcium carbonate                (21,25). In our study, Ca2 was not significantly
mineral deposits within the vesicles.                      overexpressed in tissues where the shell is
Furthermore, selected area electron diffraction            mineralized (RI and Ut); however, we noted that
confirmed the amorphous nature of calcium                  CA4 was previously identified in chicken
carbonate. Intracellular vesicles containing               eggshell proteomes (70,71,75). We observed
ACC were previously observed inside sea                    that Ca4 was expressed in duodenum, kidney
urchin epithelial cells (6,59,60). In vertebrates,         and magnum, in addition to Ut where eggshell
amorphous calcium phosphate (ACP) has been                 calcification occurs (Fig. 8). The presence of
observed in vivo inside intracellular vesicles in          CA4 protein in the uterine epithelial cells was
embryonic        mouse       osteoblasts       (61).       confirmed by immunofluorescence, and was
Extracellular vesicles with mineral were also              overabundant at the onset of shell calcification
reported for embryonic chick femur (62) and                compared to later stages. Finally, proteomic
growth plate cartilage of chicken (63,64). As an           analysis of purified UF EVs confirmed that
extension of these observations, we suggest that           CA4 is present in vesicles. CA4 is a
ACC is packaged inside vesicles when they are              glycosylphosphatidylinositol        (GPI)-linked
formed within the uterine cells. In other                  protein (76), and could be anchored to the
biomineralization models, it is known that                 outside of vesicle membranes and catalyze the
intracellular calcium is stored in the                     reversible hydration of CO2 into HCO3- at this
endoplasmic reticulum (65). However, the                   location. We propose that the CA4-catalysed
subcellular source of ACC-containing EVs                   accumulation of high levels of bicarbonate in
detected in our study remains unclear.                     proximity to the vesicular stores of Ca would
                                                           promote the formation of intravesicular ACC.
Expression of vesicular transport actors                   SLC4A7 is a sodium bicarbonate cotransporter
        The experimental evidence from this                containing         transmembrane         domains
study is summarized in a diagram of ACC                    (https://www.uniprot.org/),        which      was
vesicular transport to the mineralizing site, in           identified in matrix vesicles released from
which the main identified vesicular actors are             mineralizing human osteoblast-like cells (34).
incorporated (Fig.7). Amongst the genes that               Although SLC4A7 expression was detected in
we investigated, annexins can function as                  chicken          uterus      by        RNA-Seq
calcium channels (66-69), to enable uptake of              (https://anr.fr/Projet-ANR-13-BSV6-0007), in
the calcium ions required for intravesicular

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New model for avian eggshell formation via stabilized ACC

this study we observed that it was not                     attached to EV membrane via their
significantly overexpressed in RI and Ut..                 phosphatidylinositol 4, 5 bisphosphate-binding
          We also quantified the mRNA levels of            sites (Table 3). PDCD6IP, syntenin-1, ezrin
numerous additional vesicle markers within                 have been previously identified in bone and
uterine epithelial cells. Among the 20 vesicular           cartilage EVs (31-33).
genes highly expressed in RI and Ut, seven                          In our previous study, we proposed a
encode proteins ranked in the Top 100 of EV                role for EDIL3 and MFGE8 in targeting
protein markers by Vesiclepedia (Table S2).                vesicles containing ACC cargo to the
These seven proteins encoded by Pdcd6ip,                   mineralization site (27). EDIL3 and MFGE8 are
Hspa8, Anxa2, Ywhaz, Sdcbp, Tsg101 and                     the 6th and 34th most abundant eggshell proteins,
Anxa1 are respectively reported to rank 1st, 3rd,          respectively (71). EDIL3 was previously
5th, 13th, 19th, 39th and 43th by Vesiclepedia (39),       identified in the proteome of cartilage EVs (33).
and were previously reported as constituents of            In this study, we identified EDIL3 in purified
the avian eggshell proteome (Table S2) (70-                EVs (Table 2 and 3), and demonstrated its
73,77-86). Moreover, 12 vesicular genes                    vesicle specificity by Western blotting
(Anxa1, Anxa2, Cd82, Edil3, Hspa8, Pdcd6ip,                (Fig.5A). EDIL3 is synthesized by the tubular
Rab5a, Sdcbp, Tsg101, Vamp7, Ywhah and                     glands of the uterus (Fig.6) and could bind to
Ywhaz) encode proteins previously detected in              vesicles via its phosphatidylserine-binding
exosomes or matrix vesicles of bone cells,                 domain. In addition, EDIL3 was more abundant

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mineralizing cartilage or articular cartilage              during the initiation of mineralization (5-10 h
(Table S2) (31-34,87). The 20 genes that are               p.o., Fig.6), when massive ACC deposits are
highly expressed in oviduct segments                       detected (71). Concerning MFGE8, which is
responsible for eggshell mineralization encode             paralogous to EDIL3, we observe a high
proteins that have been incorporated into our              expression of its mRNA in RI compared to other
eggshell vesicular transport model (Fig.7; Table           tissues; only liver exhibits a higher mRNA level
S2). Some are involved in the general                      (27). MFGE8 was not detected in our proteomic
functioning of EVs such as vesicle formation               analysis of EVs, although Western blotting
and/or trafficking. Indeed, the genes Pdcd6ip,             revealed its presence in UF and to a lesser
Sdcbp, Tsg101, and Vps26a encode proteins                  extend in EVs (Fig. 5B). Altogether, these
involved in the biogenesis of EVs (32,35-38).              results strongly suggest that EDIL3 is a key
Proteins encoded by Rab5a, Rab27a, Vamp7,                  protein in EVs at the initiation stage of eggshell
Vamp3 and Vps4b participate in membrane                    biomineralization, while an EV-specific role for
fusion / vesicle release (32,35,36,38). Yhwah              MFGE8 is unlikely.
and Ywhaz encode signaling proteins (32),
while Hspa8 and Cd82 encode chaperone and                  ACC stabilization
membrane organizer proteins, respectively                           We propose two main hypotheses to
(32,36,38). Our proteomics analysis also                   explain the presence of stabilized ACC in
confirmed the presence of programmed cell                  uterine EVs, which are not mutually exclusive.
death 6-interacting protein (PDCD6IP) and                  Firstly, accumulation of ACC in the interior of
syntenin-1 (Sdcbp) in purified vesicles (Table 2           an EV may be sufficient to ensure its
and 3). Syntenin-1 (Sdcbp) is a signaling protein          stabilization, as reported in vitro for ACC
and a biogenesis factor described as an                    nanoparticles stabilized in liposomes (89,90). In
extracellular vesicle marker in Vesiclepedia               this case, the membrane coats ACC and shields
(http://microvesicles.org/extracellular_vesicle_           it from bulk water; reduced water content in the
markers). In addition, immunofluorescence for              ACC milieu is sufficient to delay crystallization
PDCD6IP revealed its presence at each stage                (91,92). Under encapsulated conditions, the
and at higher levels during the earliest stage.            initial ACC remains isolated from the aqueous
PDCD6IP is a biogenesis factor for EVs, and is             milieu and is observed to dehydrate to the more
the most frequently reported extracellular                 stable anhydrous ACC phase (90). Moreover,
vesicles marker (Vesiclepedia). Ezrin is another           magnesium and phosphate ions, which are
common EV marker, which is involved in                     present in uterine fluid and in the eggshell
plasma membrane/cytoskeleton cross-linkage                 mineral, are also reported to enhance the
during vesicle formation (39,88); it is present in         stability of ACC and to delay its crystallization
uterine fluid EVs (Table 3). The biogenesis                (91-93). Physicochemical vesicular conditions
factor, syntenin-1 (Sdcbp) and ezrin could be              could therefore be a key element for ACC

                                                       7
New model for avian eggshell formation via stabilized ACC

stabilization in uterine fluid EVs during               concentrations (107). Alpha-2-macroglobulin
eggshell biomineralization.                             (A2M), another protein identified in our study
         An alternative hypothesis incorporates         (Table 2), is an egg yolk protein able to interact
additional molecules to thermodynamically               with low-density lipoprotein, which exhibits
stabilize the transient ACC phase (26). Indeed,         calcium binding properties and consequently
previous studies have shown that organic matrix         could           interact        with         ACC
components of sea urchin spicules, coral                (https://www.uniprot.org/) (18,108). Ovomucin
skeleton or mollusk shell can stabilize ACC             (MUC), ovomacroglobulin (ovostatin, OVST)
(94-96). Moreover, ACC was observed using               and ovoglobulin G2 (TENP or OVOG2) are egg
cryo-electron microscopy inside the vesicles of         white proteins, whereas vitelline membrane
the sea urchin cells involved in spicule                outer layer protein (VMO1) is a protein
mineralization (59,60). In this context, we paid        enriched in the vitelline membrane (Table 2)
particular attention to lysozyme (LYZ) and              (109). They were previously identified in
ovalbumin (OVA) that were identified in our             eggshell (70,71), but their role related to the
proteomic study (Table 2 and 3), and were               calcification process is unknown. Clusterin
previously reported in chicken eggshell                 (CLU) is a chaperone protein previously
(70,75,77-79,81,97) and shown to modify                 identified in eggshell and proposed to prevent
calcium carbonate precipitation and calcite             the premature aggregation and precipitation of
crystal morphology in vitro (98,99). The role of        eggshell matrix proteins during calcification

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LYZ in the stabilization of ACC has been                (110). Aminopeptidase N (AMPN) and Loc
explored but remains controversial. Only high           771972 (OCX25) are a protease and protease
concentrations of LYZ alter the morphology of           inhibitor, respectively (15,111), which were
calcite crystals in vitro, and LYZ is ineffective       also detected in vesicles (Table 2). Protease and
in the stabilization of ACC particles (99).             protease inhibitors could potentially control the
However, metastable ACC was obtained in                 calcification process, either by degrading
vitro in the presence of chicken egg white              proteins or by modifying the processing of
lysozyme C (100,101). Lysozyme markedly                 protein maturation (71). Prominin-1-A
decreased the average diameter of metastable            (PROM1), a protein involved in lipid
ACC particles and promoted a network of                 metabolism was also present in vesicles but its
associated particles that incorporated the              role is not yet defined. Finally, nine EV proteins
protein into the precipitate (100). Additionally,       are partial immunoglobulins, with unknown
lysozyme-ACC particles transform exclusively            functions in vesicles or in shell mineralization.
into the crystalline calcite polymorph
(100,101).                                              Conclusions
         OVA is an abundant eggshell matrix                      In the present study, we provide
protein, and its role in calcium carbonate              evidence for the first time in vertebrates of a
formation and ACC stabilization has been                vesicular transport pathway for delivery of
investigated (98,102-105). Calcium binds to             stabilized amorphous calcium carbonate
OVA and this accumulation creates a nucleation          mineral particles. We used multiple approaches
center for mineral formation (105). Calcium             to confirm predictions based on this model.
ions are bound to the protein by complexation           TEM coupled to spectroscopic analyses
through acidic groups leading to protein                demonstrated several phases of EV formation at
rearrangement (97). The calcium cations are the         uterine cells, as well as the presence of EVs
starting points for the subsequent formation of         containing ACC mineral deposits in the UF. The
ACC nuclei, which then undergo a series of              major actors involved in this vesicular transport
phase transitions to the stable crystalline             were organized into a comprehensive schema
polymorphs (104). OVA stabilizes the unstable           (Fig. 7), which incorporates EVs and
calcium carbonate phases in vitro (105,106).            constituent proteins in the process of shell
Therefore, the presence of OVA in vesicles will         mineralization. EDIL3 is a targeting protein that
contribute to the formation of metastable ACC           is proposed to address vesicles for delivery of
(Table 3, Fig.7).                                       ACC to the mineralization site. Calcium
         We also identified about 20 additional         accumulation by vesicles is promoted by
proteins in UF EVs by proteomics (Table 2).             annexins 1, 2 and 8, whereas carbonic
Ovotransferrin (OVOT) can modify calcite                anhydrase 4 catalyzes bicarbonate formation
crystal morphology in vitro at low                      from CO2. Moreover, our study identified and

                                                    8
New model for avian eggshell formation via stabilized ACC

characterized several additional proteins that          whereas mid-shaft tibial bones (B) were
could contribute to the stabilization of ACC            collected at 18 h p.o from 90 week old hens.
inside vesicles, under favorable physical and           Oviduct epithelium samples were obtained by
chemical conditions.                                    scraping the inner surface. Mid-shaft tibial bone
                                                        samples were cleaned of muscle but were not
Experimental procedures                                 flushed of marrow; duodenum tissues were
Ethical statement and housing                           directly cleaned by immersion in physiological
         All animal-handling protocols were             saline. Liver was collected from the right lobe.
carried out in accordance with the European             After that, tissues were directly immersed in
Communities Council Directives concerning               liquid nitrogen and then stored at -80 °C in cryo-
the practice for the care and use of animals for        tubes until RNA or protein extraction.
Scientific Purposes and the French Ministry on                   Small pieces of uterine wall (0.5 cm2)
Animal experimentation, and were performed              were also sampled at 16 h p.o. from 40 week old
under the supervision of authorized scientists          hens for electron microscopy, and were fixed by
(authorization #7323, delivered by the French           incubation at room temperature for 24 h in 4%
ministry of Education and Research). Birds              paraformaldehyde, 1% glutaraldehyde in 0.1 M
were kept in the experimental unit PEAT 1295            phosphate buffer (pH 7.3). Fixed tissues were
of INRA, which has permission to rear birds and         stored at 4°C until preparation of sections.
to perform euthanasia of experimental animals                    For immunofluorescence analysis,
(decree N° B37-175-1 of August 28th 2012

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                                                        small pieces (2 cm x 0.4 cm) of the uterus at
delivered by the “Préfécture d’Indre et Loire”          each stage (5 h, 6 h, 7 h, 10 h and 16 h p.o.),
following inspection by the Direction of                from 25 hens at 40 week old, were embedded in
Veterinary Services). Our experimental                  O.C.T.      (Optimal     cutting     temperature)
protocol was approved by the ethical committee          compound         mounting        medium        for
“Comité d’éthique de Val de Loire, officially           cryosectioning (VWR, Radnor, USA) using
registered under number 19 of the French                molds and direct immersion in isopentane
National Ethics Committee for Animal                    cooled by liquid nitrogen. The blocks of O.C.T.
Experimentation” under agreement number                 containing uterus pieces were stored at -80 °C
#16099-015902.                                          until cryosectioning.
         Mature brown egg-laying hens (ISA-                      UF was collected as previously
Hendrix, 40 and 90 weeks old at sampling) were          described (20). Briefly, egg expulsion was
kept in individual cages furnished with                 induced by injection of 50 µg prostaglandin-
automatic equipment for recording of the                F2α in the alar vein of 40 week old hens, either
oviposition (egg-laying) times. Animals were            at 9 h or 16 h p.o. Following egg expulsion, UF
fed layer mash ad libitum and exposed to a 14 h         was collected directly by gravity in a plastic
light / 10 h darkness cycle. Following collection       tube placed at the entrance of the everted
and removal of egg contents, the eggshells were         vagina. A portion of the fluid was promptly
thoroughly washed with water, air dried and             pipetted and deposited in liquid nitrogen to
stored at -20°C.                                        obtain 50 µL beads of uterine fluid for storage
Tissue and uterine fluid collection                     at -80 °C until use for electron microcopy. The
         Thirty-two brown laying hens (ISA-             remaining UF was diluted in phosphate
Hendrix, 26 and 6 at 40 and 90 weeks old,               buffered saline (PBS, 4 v: 1 v) in cryotubes
respectively) were sacrificed by lethal                 maintained on ice and immediately used for
intravenous       injection    of     Dolethal®         extracellular vesicle isolation.
(Vetoquinol, Magny-vernois, France) at either           Messenger RNA levels
the initial phase of eggshell mineralization (5-7               Total RNA was extracted from tissues
h p.o.), the onset of the active mineralization         harvested at 10 h p.o. (except bone, which was
phase (10 h p.o.) or during the linear growth           extracted at 18 h p.o.), and treated as previously
phase of rapid mineralization (16-18 h p.o.).           described (27). Briefly, the concentration of
         For RT-PCR and Western blotting                each RNA sample was measured at 260 nm with
analyses, various tissues (Duodenum, D;                 NanoDrop™ and their integrity was assessed on
Kidney, K; Liver, L) and oviduct regions                1.5 % agarose gels. Total RNA samples (1 µg)
(Magnum, Ma; White isthmus, WI; Red                     were reverse-transcribed using RNase H-
isthmus, RI and Uterus, Ut) were collected from         MMLV reverse transcriptase (Superscript II,
40 week old hens sacrificed at 10 h p.o.,

                                                    9
New model for avian eggshell formation via stabilized ACC

Invitrogen, Carlsbad, California) and Oligo                           The Ca4 mRNA was amplified by end-
(dT)™       primers         (Invitrogen,     Carlsbad,        point PCR in each oviduct segment and tissues
California). Primers for the Edil3, Mfge8, Ca4,               using DreamTaq PCR Master Mix 2X
the 31 vesicular genes and the eight                          (ThermoFisher Scientific, Waltham, USA) and
housekeeping genes (B2m, Eif3i, Gapdh, Gusb,                  Mastercycler gradient (Eppendorf, Hambourg,
Stag2, Tbp, Sdha, and Ppia) were designed from                Germany). After 3 min of denaturation, 35
Gallus gallus gene sequences (Table S3), using                cycles were used (95 °C, 30 s; 60 °C, 30 s; 72
Primer-BLAST on National Center for                           °C, 30 s) to amplify PCR products, followed by
Biotechnology Information (NCBI), and were                    a final elongation for 15 min. PCR products
synthesized (Eurogentec, Lièges, Belgium).                    were then mixed with Trackit cyan/orange
Primer efficiencies were evaluated by RT-                     Loading buffer 6 X (ThermoFisher Scientific,
qPCR using LightCycler® 480 SYBR Green I                      Waltham, Massachusetts), loaded on a 2.5 %
Master and LightCycler® 480 instrument II                     agarose gel and migrated for 30 min at 100 V
(Roche, Bâle, Switzerland). Messenger RNA                     using the Mupid-One electrophoresis system
level was quantified using the Biomark                        (Dominique Dutscher, Issy-les-mouligneaux,
microfluidic system, in which every sample-                   France). The Trackit 100-bp DNA ladder was
gene combination is quantified using a 96.96                  used (ThermoFisher Scientific, Waltham,
Dynamic Array™ IFCs (BMK-M-96.96,                             Massachusetts) to determine the size of PCR
Fluidigm®, San Francisco, California) at the                  products. Imaging was performed using the

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GeT-GenoToul platform (Toulouse, France).                     Syngene GeneGenius Gel Light Imaging
Samples were pre-amplified according to the                   System (Syngene, Cambridge, UK).
manufacturer’s specifications. PCR was then
performed using the following thermal                         Extracellular vesicle isolation from uterine
protocols: Thermal Mix (50 °C, 2 min; 70 °C,                  fluid and extraction of soluble eggshell matrix
30 min; 25 °C, 10 min), Hot Start (50 °C, 2 min;              proteins
95 °C, 10 min), 35 PCR cycles (95 °C, 15 s; 60                         EVs were isolated from uterine fluid
°C, 60 s), and Melting Analysis (60 °C, 30 s; 95              harvested at 9 h and 16 h p.o., using previously
°C, 1 °C/3 s). Real time quantitative PCR results             described methodology (112). Briefly, UF
were analyzed using Fluidigm Real-time PCR                    diluted in PBS was centrifuged at 100 x g for 15
analysis                 software              v.4.1.3        min and then at 12, 000 x g for 15 min to remove
(https://www.fluidigm.com/software).               Six        cell debris. Two successive ultracentrifugation
biological replicates and two technical                       steps at 100, 000 x g (Beckman Coulter L8-
replicates were carried out for each tissue.                  70M, Beckman Coulter, Brea, California) were
GenNorm software (https://genorm.cmgg.be/)                    then performed during 90 min to pellet the EVs.
was used for validation of housekeeping gene                  Aliquots of the supernatants corresponding to
stability. The normalized quantities were                     UF without EVs were sampled and stored at -20
calculated using the following formula: (gene                 °C. The pellet was suspended in 50 µL of PBS
efficiency (ctcalibrator-ctsample))/geometric average         and stored at -20° C until transmission electron
quantity of the eight housekeeping genes.                     microscopy (TEM), nanoLC-MS/MS and
Normalized quantities were compared between                   Western blot analysis.
various measured tissues and oviduct segments                          Soluble eggshell proteins (SEP) from
using one-way ANOVA followed by Tukey                         normally laid eggs were extracted as previously
pairwise test analysis on Minitab® 18 software                detailed (107). Briefly, eggshell pieces with
(http://www.minitab.com/fr-fr/). A p-value <                  eggshell membranes were immersed in 154 mM
0.05 was selected as the threshold for                        NaCl containing protease inhibitors (2.5 mM
significance between different groups. The                    benzamidine-HCl, 50 mM ε-amino-n-caproic
heatmap of the mRNA levels (Row-Z score) in                   acid, 0.5 mM N-ethymaleimide, and 1 mM
different tissues for Edil3, Mfge8 and the 31                 phenylmethylsulfonyl fluoride), and then
vesicular genes was computed using the                        ground into a fine powder. Eggshell powders
heatmap.2 function from gplots packages of                    were fully demineralized by immersion in 20 %
Rstudio                 software              1.1.456         acetic acid, and the resulting suspensions were
(https://www.rstudio.com/). Row and column                    dialyzed (cut off 3,500 Da; dialysis membrane
clustering were performed using pearson-                      Spectra/Por™,       ThermoFisher      Scientific,
ward.D2 methods and spearman-Ward.D2                          Waltham,          Massachusetts)          against
methods, respectively.                                        demineralized water for 24 h at 4 °C and

                                                         10
New model for avian eggshell formation via stabilized ACC

lyophilized. Powdered samples were incubated             blocking step was performed with 1X PBS, 3 %
overnight at 4 °C in 4 M guanidine-HCl, 5 mM             BSA for 20 min at room temperature. Sections
benzamidine-HCl, 0.1 M ε-amino-n-caproic                 were then incubated in 1X PBS, 3 % BSA with
acid, 10 mM EDTA, 50 mM sodium acetate and               diluted primary antibodies (anti-ANXA1, anti-
1 mM phenylmethylsulfonyl fluoride, and then             ANXA2, anti-ANXA8, anti-CA4, anti-EDIL3
dialyzed (cut off 3, 500 Da) against 0.5 M               or anti-PDCD6IP, Table S4). Sections were
sodium acetate pH 7.4 for 24 h at 4 °C, followed         washed 3 times at room temperature for 5 min
by centrifugation at 2, 000 x g for 10 min at 4          in 1X PBS. Negative controls were prepared
°C. The resulting SEP (supernatants) were                without primary antibody. Sections were
stored at −20 °C.                                        washed 3 times for 5 min at room temperature
                                                         in 1X PBS. Then, according to the primary
Electrophoresis and Western blot analyses                antibody, sections were incubated with either
        Protein concentrations were determined           goat anti-rabbit secondary antibody (IgG, H+L),
using the BioRad DC Protein Assay kit II                 AlexaFluor® 488 (1: 1, 000; ThermoFisher
(BioRad, Marnes-la Coquette, France) in                  Scientific, Waltham, Massachusetts) or goat
accordance with manufacturer’s instructions              anti-mouse IgG1 cross-adsorbed secondary
and using bovine serum albumin (BSA; Sigma-              antibody, AlexaFluor® 555 (1: 1, 000;
Aldrich, Saint-Quentin Fallavier, France) as             ThermoFisher          Scientific,     Waltham,
standard. Fifteen micrograms of each sample              Massachusetts) (Table S4) in 1X PBS, 1 % BSA
were diluted into Laemmli sample buffer

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                                                         for 1 h at room temperature. All
(5v:1v) and boiled for 5 min. Protein samples            immunolabeling was performed on duplicate
were then separated on 12 % polyacrylamide               sections. Sections were washed three times for
gels (Mini-Protean II electrophoresis cell,              5 min at room temperature and then mounted
BioRad, Marnes-la-Coquette, France) and                  using Fluoroshield™ with DAPI histology
transferred to 0.2 µm nitrocellulose blotting            mounting medium (Sigma-Aldrich, Saint-
membrane (GE Healthcare, Little Chalfont,                Quentin Fallavier, France). Sections were
UK) for Western blot analysis. Briefly,                  observed with Axioplan II Fluorescence
membranes were washed 5 min in Tris buffered             Microscope (Zeiss, Oberkochen, Germany) and
saline (TBS; 50 mM Tris-HCl, 150 mM NaCl,                images were acquired using an INFINITY
pH 7.4), blocked with the Odyssey® blocking              microscope camera (Lumenera, Nepean,
buffer (LI-COR, Bad Homburg, Germany) in                 Canada).
TBS (1v:1v), and then incubated for 3 h in the
blocking solution (Odyssey® blocking buffer              Proteomics analysis of uterine fluid
1v: TBS 1v) containing 0.1% Tween-20 and                 extracellular vesicles
anti-EDIL3 (1: 1, 000) or anti-MFGE8                             Sixty micrograms of purified EVs
antibodies (1: 1, 000) (Table S4). Membranes             resuspended in PBS were diluted into 2 % SDS
were sequentially washed for 5 min in TBS, 0.1           Laemmli sample buffer (5v:1v) and boiled for 5
% Tween-20, and then incubated for 1 h in the            min. EV proteins were electrophoresed on a
blocking solution containing 0.1 % Tween-20              12.5 % polyacrylamide gel (50V, 30 min) to
and AlexaFluor® 680 goat anti-rabbit (H+L)               obtain a single band (Mini-Protean II
secondary antibody (1: 20, 000; ThermoFisher             electrophoresis cell, BioRad, Marnes-la-
Scientific, Waltham, Massachusetts) (Table               Coquette, France). The gel was stained with
S4). Finally, membranes were washed three                Coommassie Blue and the single band of
times in TBS containing 0.1 % Tween-20 and               proteins was excised and subjected to in-gel
twice in TBS. Immuno-reactive bands were                 digestion with bovine trypsin (Roche
revealed using the Odyssey® imaging system               Diagnostics GmbH, Mannheim, Germany) as
(LI-COR, Bad Homburg, Germany) at 700 nm.                previously described by Marie et al. for analysis
                                                         by nanoscale liquid chromatography–tandem
Immunofluorescence of uterine tissue                     mass spectrometry (nanoLC–MS/MS) (84).
          Serial sections (10 µm) were prepared          MS/MS ion searches were performed using
from uterus embedded in O.C.T. (harvested at 5           Mascot search engine version 2.6 (Matrix
h, 6 h, 7 h, 10 h and 16 h p.o.) using the CM3050        Science, London, UK) via Proteome Discoverer
S cryostat (Leica, Wetzlar, Germany). Sections           2.1 software (ThermoFisher Scientific, Bremen,
(stored at -80 °C until use) were thawed for 30          Germany) against the NCBIprot_chordata
min at room temperature and rehydrated during            database (July 2018). The search parameters
5 min in 1X PBS before immunolabeling. The

                                                    11
New model for avian eggshell formation via stabilized ACC

included trypsin as a protease with two allowed          Transmission electron microscopy and
missed cleavages, carbamidomethylcysteine,               energy-dispersive X-ray spectroscopy analyses
methionine oxidation and acetylation of protein          of uterus and uterine fluid
N-termini as variable modifications. The                          Fixed pieces of uterus were washed in
tolerance of the ions was set to 5 ppm for parent        PBS and post-fixed by incubation with 2 %
and 0.8 Da for fragment ion matches. Mascot              osmium tetroxide for 1 h. Samples were then
results obtained from the target and decoy               fully dehydrated in a graded series of ethanol
databases searches were subjected to Scaffold            solutions and embedded in Epon resin, which
software (v 4.8.8, Proteome Software, Portland,          was polymerized by heating from 37 °C to 60
USA) using the protein cluster analysis option           °C. Ultra-thin sections (50–70 nm thick) of
(assemblage of proteins into clusters based on           these blocks were obtained with a LEICA
shared peptide evidence). Peptide and protein            Ultracut UCT ultramicrotome (Leica, Wetzlar,
identifications were accepted if they could be           Germany). Sections were stained with 5 %
established at greater than 95.0 % probability as        uranyl acetate, 5 % lead citrate and observations
specified by the Peptide Prophet algorithm and           were made with a TEM-1400 Plus electron
by the Protein Prophet algorithm, respectively.          microscope (JEOL, Tokyo, Japan).
Protein identifications were accepted if they                     UF harvested at 16 h p.o. and stored as
contained at least two identified peptides.              frozen beads, was deposited on a TEM grids (C
                                                         coated) and then dried for 1 min. Excess UF was
Transmission electron microscopy and

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                                                         then absorbed with filter paper, stained with
electron energy loss spectroscopy analyses of            uranyl acetate 2 % and the grid was immediately
uterine fluid                                            observed with a TEM-1400 Plus electron
         Frozen beads of UF harvested at 16 h            microscope (JEOL, Tokyo, Japan). EVs were
p.o were deposited on a Cu TEM grids and dried           observed under the TEM (BF/DF detector) and
for 1 min. Excess UF was then absorbed with              energy-dispersive X-ray spectroscopy (EDS;
filter paper and the grid was immediately                Oxford 65 mm2 detector) analysis was
observed with the TEM-ZEISS LIBRA 120                    performed on EVs in order to detect elemental
instrument (ZEISS, Oberkochen, Germany).                 calcium, carbon and oxygen (AZtec software).
EVs were localized under the TEM and electron            Selected area electron diffraction (SAED)
energy loss spectroscopy (EELS) spectra from             patterns were registered to determine the
270 to 410 eV and from 460 to 600 eV were                crystallinity of mineral deposits observed wthin
acquired (Table S1). Mapping of elemental                EVs.
calcium (345-355 eV) and carbon (280-290 eV)
were performed to determine the spatial                  Data Availability
distribution of CaCO3 and organic carbon in              All data are contained within the manuscript.
EVs.
Acknowledgements: The authors are grateful to the experimental unit (PEAT, INRAE, 2018. Poultry
Experimental Facility, doi: 10.15454/1.5572326250887292E12) for the care of birds, to the GeT-
GenoToul platform Toulouse for performing real time quantitative PCR with the Biomark Fluidigm
system and to the Centro de Instrumentación Científica de la Universidad de Granada for electron
microscopy and spectroscopic analysis. The authors acknowledge Pierre-Ivan Raynal and Sonia
Georgeault from the IBiSA electron microscopy platform of the “Université François Rabelais” of Tours
for their assistance. The authors also acknowledge Nadine Gérard, Cindy Riou and Agostinho Alcantara-
Neto from UMR “Physiologie de la Reproduction et des Comportements” (INRAE Centre Val de Loire)
for their help in extracellular vesicle isolation. The authors wish to thank the Université François
Rabelais de Tours and the Région Centre Val de Loire for financial support of Lilian Stapane’s doctoral
studies. MTH acknowledges funding from NSERC (RGPIN-2016-04410) and is grateful to Le
STUDIUM for support during the preparation of this manuscript. He is a Le STUDIUM Research
Fellow, Loire Valley Institute for Advanced Studies, Orleans-Tours, and BOA, INRAE, Centre Val de
Loire, Nouzilly, France.
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this
article.

                                                    12
New model for avian eggshell formation via stabilized ACC

Author contributions: LS was involved in designing and planning the study. He performed
experiments and analyses, interpreted data and statistical analyses and wrote the original draft of the
paper. NLR was involved in the conceptualization of the study, the experimental design and the
interpretation of data and contributed to the writing of the paper. JE, ABRN, VL, LCS performed
experimental procedures and interpreted data. MTH was involved in the conceptualization and the
writing of the paper. JG conceived the strategy, designed and experiments, interpreted data and statistical
analyses and was involved in the writing of the paper. He acquired funding and supervised the study.
All authors have read and approved the final manuscript.

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