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
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
1
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
2
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
3
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
4
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
5New 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
6New 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
7New 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
Downloaded from http://www.jbc.org/ by guest on November 25, 2020
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
8New 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.,
9New 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
10New 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
11New 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.
12New 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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