3. Gene symbol of proteins (ionic transporters, shell matrix
proteins) are described in Tables 32.1 and 32.2.
1,25(OH)2D3,25-dihydroxyvitamin D3or 25(OH)D 25
hydroxyvitamin D3 Metabolites of vitamin D.
32.1 Introduction
Birds are oviparous and produce a cleidoic egg with its
internal environment almost totally isolated from the exte-
rior. This reproductive cell is composed of an oocyte sur-
rounded by nutritional reserves. The unfertilized chicken
egg is consumed worldwide because of its low price and
high-nutritional value. It contains a large diversity of
nutrients (protein, energy, vitamins, and minerals) that are
largely sufficient for the human diet with the exception of
calcium and vitamin C (Rehault-Godbert et al., 2019). Eggs
also possess an ideal profile of balanced amino acids and
contain large amounts of unsaturated fatty acids. The egg is
similar between different species of birds: a yolk sur-
rounded by an egg white, eggshell membranes (ESMs), and
the eggshell. The egg is a package containing all compo-
nents needed for the development of the embryo, with a
variety of protective systems against physical and microbial
attacks. The egg white contains numerous antimicrobial
proteins and the eggshell protects the contents of the egg
from mechanical insults. In addition, shell porosity regu-
lates the exchange of water and gases during extrauterine
development of the chick embryo; moreover, the eggshell is
also a calcium reserve that supports embryonic bone
development. The shell mineral structure is one of the most
impressive terrestrial adaptations in amniotes, which allows
embryonic development in the desiccating nonaquatic
environment. Shell mineralization takes place in the lumen
of the uterus, which secretes an acellular milieu, the uterine
fluid that contains all necessary ionic and organic pre-
cursors. Its particularities, as compared to bone or teeth, are
the material [calcium carbonate (CaCO3) instead of calcium
phosphate] and the absence of cell-directed assembly dur-
ing mineralization. The ESMs play a crucial role by
controlling the initial mineralization, which occurs upon
organic cores on its outer surface.
In the laying chickens, eggshell formation takes place
daily in the uterus and is one of the most rapid biominer-
alization processes known. To provide about 6 g of shell as
CaCO3, the hen exports each day 2.4 g of Ca corresponding
to 10% of her total body calcium (Sauveur and de Reviers,
1988; Nys and Guyot, 2011) and in 1 year of egg pro-
duction, the modern pedigree hen exports more than her
body weight as eggshell. The intensity and discontinuity of
Ca secretion challenges calcium homeostasis in hens;
however, birds develop physiological adaptations in the
intestine, bone, and uterus upon sexual maturity. Moreover,
there is a daily cycle in these tissues during shell formation
that provides the necessary Ca and bicarbonate. Two weeks
before the onset of egg production, hens establish a calcium
“reservoir”dthe medullary bone, display a largely
increased intestinal Ca retention and develop the secondary
reproductive organ, the oviduct. The spatiotemporally
regulated process of egg formation takes place in special-
ized segments of the oviduct following yolk ovulation
(Figure 32.1): secretion of the vitelline membrane compo-
nents in the infundibulum, secretion of albumen in the
magnum, ESM deposition in the isthmus, and eggshell
mineralization in the uterus (Sauveur and de Reviers, 1988;
Nys and Guyot, 2011). The uterus secretes large amounts of
Ca2þ
and HCO3
ions to form the shell (Hurwitz, 1989a;
Nys, 1993; Bar, 2009). This daily export of calcium causes
a decrease in plasma calcium, which stimulates, through
Ca2þ
-sensing receptors (CaSRs), the synthesis and secre-
tion of calcium-regulating hormones: mainly parathyroid
hormone (PTH) and 1,25-dihydroxyvitamin D3, which
together influence Ca flux by acting on bone resorption and
intestinal absorption (Wasserman, 2004; Christakos, 2014,
2019). Numerous classical physiological studies have
explored the regulation of uterine ionic transfer, without
demonstration of a direct effect of these hormones upon
avian Ca metabolism (Bar, 2008, 2009; Nys and Le Roy,
2018). However, during the past 20 years, transcriptomic
and proteomic in vivo analyses have provided detailed in-
formation on the proteins involved in the mechanisms of
ion supply and of shell mineralization. The chicken con-
stitutes an excellent model because the spatial and temporal
sequence of egg formation is well known, and the oviduct
provides experimental access to tissues that are specific to
particular functions.
OVULATION (0 h)
OVIPOSITION (24 h)
Magnum (4 h)
Egg white deposion
Isthmus (1 h)
Eggshell membranes deposion
Terminal phase
22-24 h
Uterus (19 h)
Eggshell formaon
Inial phase
5-10 h
Growth phase
10-22 h
FIGURE 32.1 Spatiotemporal formation of the egg. Schematic drawing
of the egg passage through the oviduct, timing and stage of shell
formation.
814 PART | V Endocrine theme
4. Indeed, the components of each egg compartment are
produced sequentially (Sauveur and De Reviers, 1988; Nys
and Guyot, 2011). The liver synthesizes the egg yolk
components that are exported to the ovary. Following
ovulation, the largest ovarian follicle releases a mature
ovum into the oviduct. Specialized parts of the oviduct
successively synthesize and secrete: the constituents of the
outer vitelline membrane which surround the yolk, the egg
white, the shell membranes, and the eggshell. This temporal
sequence is controlled by the daily cycle of steroid and
pituitary hormones. In hens, it is possible to compare the
expression of genes between specific segments of the
oviduct at well-defined stages of egg formation in order to
obtain insight into genes involved in specific functions
associated with the formation of an egg component.
Comparison of proteomic profiles of egg compartments or
uterine fluid sampled at well-defined periods of egg for-
mation also permit the identification of proteins involved in
mechanisms of ionic transport, eggshell mineralization, and
their regulation. These approaches have revealed in detail
the mechanisms of Ca transfer at the intestinal and uterus
level, identification of novel mechanisms of uterine Ca
secretion (vesicular transport), as well as, identification of
the matrix proteins involved in the control of eggshell
mineralization. This novel information will be presented in
this chapter. Transcriptomic and proteomic approaches also
allow exploration of the regulation of these processes or
highlight novel hormone pathways, such as fibroblast
growth factor 23 (FGF23) acting on phosphate metabolism.
Such studies are mainly qualitative but have allowed a
myriad of protein candidates involved in ionic transport and
shell mineralization to be identified. More quantitative and
functional analyses are needed to hierarchize the candidates
and to understand different mechanisms involved in shell
formation. This chapter aims to update information in these
areas and to underline the novel knowledge that has
accrued on the proteins involved in providing and in
building the shell material; however, there needs to be a
better understanding of their physiological regulation.
32.2 Structure, composition, and
formation of the eggshell
32.2.1 Structure and composition
The shell structure is similar for different species of birds
and shares the same mineral component, namely the
trigonal phase of CaCO3 known as calcite, which is the
most stable CaCO3 polymorph at room temperature
(Hamilton; 1986; Solomon, 1991; Nys et al., 1999; Hincke
et al., 2012). In the shell ultrastructure, up to six layers can
be distinguished (Figure 32.2). The inner part of the
eggshell comprises two shell membranes consisting of
interlacing protein fibers. The mineral portion is anchored
on organic rich structures, the mammillary bodies, located
at the surface of the outer shell membrane fibers. These
structures have a strong calcium binding capacity and act as
nucleation sites for calcite crystal formation during the
initial stages of eggshell mineralization (Fernandez et al.,
2001). Calcite crystals grow radiating away from
mammillary bodies and forming inverted cones (mammil-
lary layer) that fuse at their bases and continue growing
outward to form a compact zone called the palisade layer.
The palisades consist of juxtaposed irregular columnar
units of calcite crystals that became larger toward the
eggshell surface, with diameters ranging between 60 and 80
microns. The palisade layer is around 200 mm thick in
chicken eggs, corresponding to about two-thirds of the
eggshell thickness and has the largest contribution to
eggshell mechanical properties. It is completed by a thin
vertical crystal layer where the crystallites are aligned
perpendicular to the shell surface which can be visualized
in thin sections of eggshell viewed under an optical
microscopy with crossed polarizing filters (Figures 32.3
and 32.4).
The main ultrastructural characteristics (columnar
structure) and mineralogical composition (calcite) of the
eggshell are constant, across all avian species, but there is a
notable variability in the eggshell microstructure charac-
teristics (size, shape, and orientation of the crystals of
calcite) between species and even within the same specie
depending on different factors (Panheleux et al., 1999;
Ahmed et al., 2005; Rodriguez-Navarro et al., 2007).
FIGURE 32.2 Left: Scanning electron micrograph (SEM) (shell thick-
ness is 300 mm)) of a cross-fractured hen eggshell showing the different
layers. Right: SEM of mammillary layer, palisade layer, and upper part of
the shell (Nys et al., 2001).
Mechanisms and hormonal regulation of shell formation Chapter | 32 815
5. The eggshell ultrastructure and microstructure are respon-
sible for the exceptional mechanical properties of eggshell
(in chickens, the egg breaking strength is 35N for a mean
thickness of 0.33 mm). Changes in these characteristics
have a strong effect on eggshell mechanical properties.
A remarkable example is the Guinea fowl eggshell
(Panheleux et al., 1999; Le Roy et al., 2019). Its inner part
is made of calcite crystal units arranged vertically as in
chickens. However, the outer zone has a more complex
microstructural arrangement made of very smaller intri-
cately interlaced calcite crystals with varying orientation
(Figure 32.3). These characteristics of the Guinea fowl
eggshell confer upon its superior mechanical properties
compared to the eggs of other birds.
The cuticle, an organic layer, is deposited on the surface
of the eggshell; it contains a large proportion (2/3) of the
superficial pigments (Nys et al., 1991). The inner cuticle
contains a thin layer of hydroxyapatite crystals (Dennis
et al., 1996). About 10,000 respiratory pores penetrate the
hen eggshell (200 pores/cm2
), which are plugged by the
cuticle. They allow and control the exchange of water and
metabolic gases during the extrauterine development of the
chick embryo while impeding bacterial penetration through
the shell and preventing contamination of the egg contents.
The ESMs are composed of disulfide-rich protein fibers
(w10% cysteine) that are extensively cross-linked by
irreversible lysine-derived crosslinks of desmosine and
isodesmosine. Collagen was suggested to be present
because of identification of hydroxylysine, the observation
of digestion of ESMs by collagenase, and finally by
immunochemistry using antibodies against type I, V, and X
collagen (Wong et al., 1984; Arias et al., 1997; Wang et al.,
2002). However, the amino acid composition of the shell
membranes largely differs from that of collagenous tissues,
suggesting that collagen is not predominant. In fact, a
combination of proteomics and transcriptomics approaches
have revealed that a major ESM component is a cysteine-
rich eggshell matrix protein (abbreviated CREMP), whose
sequence displays similarity to spore coat protein SP75 of
cellular slime molds (Kodali et al., 2011). The structural
proteins CREMP, collagen X, and fibrillin-1 are highly
overexpressed in the white isthmus segment of the oviduct,
which is responsible for the synthesis and secretion of the
ESM constituents. CREMP contains around 14% cysteine,
in contrast to collagen X (a-1) which is only 0.2% cysteine,
suggesting that CREMP could account for the relatively
high-cysteine content of eggshell (Du et al., 2015). Prote-
omics investigations suggest that the most abundant ESM
proteins are CREMP, collagen X, lysyl oxidaseelike 2
(LOXL2) and lysozyme, with the remaining approximately
25% constituted by more than 500 proteins (Ahmed et al.,
2017, 2019a, 2019b).
A B C
FIGURE 32.3 Cross-section of eggshell viewed in cross-polarized light photomicrographs showing the orientation of calcite crystals in the eggshells
from hen (A), turkey (B), and guinea fowl (C). Note the presence of the thin vertical crystal layer at the top of the turkey shell (B) and the presence of
interlaced calcite crystals in the upper palisade layer of the guinea fowl shell (C). By courtesy of Juan Manuel Garcia-Ruiz, Laboratorio de Estudios
Cristalograficos, Instituto Andaluz de Ciencias de la Tierra, Granada, Spain; Panheleux et al. (1999).
816 PART | V Endocrine theme
6. 32.2.2 Kinetics and site of shell membranes
and shell formation
ESM fibers are synthesized and secreted by glandular cells
of the white isthmus 4 h after yolk ovulation (Nys et al.,
1999, 2004). The organic components of mammillae
(nucleation sites) and the first crystals are laid down on the
external shell membranes in the distal red isthmus 5 h after
ovulation. The progressive hydration of the egg albumen
swells the forming egg, creating its ovoid shape and
allowing close contact with the uterine wall about 10 h after
ovulation. Active secretion of calcium, carbonate, and
organic precursors over the following 12e14 h contribute
to the rapid and linear deposition of the shell mineral,
which ends with cuticle secretion about 1.5 h before
oviposition (egg expulsion). Eggshell formation is the
longest step of egg formation as it lasts about 20 h if the
initial phase of shell nucleation is included; it is initiated at
4.5 h after ovulation and ends 1.5 h before oviposition.
Shell formation occurs in three periods, the nucleation
phase (5e10 h after ovulation), the rapid deposition of shell
material (10e22 h after ovulation), and the termination of
mineralization (21e23 h after ovulation).
32.3 Mineral supply: a challenge for
calcium metabolism
No calcium storage occurs in the uterus before the initiation
of shell formation (Sauveur and de Reviers, 1988; Nys
et al., 1999). Calcium is directly provided by the ionic
blood calcium. The amount needed to form a shell (2 g of
Ca in chicken) is very large and the pool of blood ionic
calcium in hens laying more than 320 eggs/year must be
provided at a rate equivalent to its renewal every 12 min.
The laying chicken exports, during 1 year of egg produc-
tion, more than her body weight as eggshell. Calcium is
initially provided by the hen’s diet. During eggshell calci-
fication, about two thirds of the Ca deposited in the uterus
is directly supplied by the hen’s diet, while one-third
(30e40%) is mobilized from bone. Bone calcium mobili-
zation is necessary because there is a desynchronization
between food intake during the day and egg formation,
which mainly takes place during the night. Provision of
high-dietary level of Ca (3.5%), in the form of large par-
ticles of CaCO3, provides sufficient intestinal Ca during the
night to reduce the degree of bone mobilization for shell
formation. Conversely, a hen fed a diet which is low in Ca
FIGURE 32.4 Upper part: Cross-section of eggshell viewed in cross-polarized light. Photomicrographs showing the orientation of calcite crystals in the
eggshells from hen (A, larger randomly oriented crystals), emu (B, randomly oriented microcrystals), and ostrich (C, preferred orientation). Lower part:
2D-XRD patterns of eggshell of hen (D), emu (E), and ostrich (F) analyzing crystal orientation and size (Rodriguez-Navarro et al., 2002).
Mechanisms and hormonal regulation of shell formation Chapter | 32 817
7. will mobilize up to 58% of the bone calcium. This cycle of
daily resorption of bone is facilitated in hens by the pres-
ence of medullary bone. Two weeks before the onset of egg
production, immature hens develop a novel and easily
mobilized calcium “reservoir,” the medullary bone. In
addition, sexually mature laying hens largely increase their
capacity to absorb Ca in the intestine under the control of
the active metabolite of vitamin D (Hurwitz, 1989a; Nys,
1993; Bar, 2008; Nys and Le Roy, 2018) resulting in a
threefold increase in intestinal Ca retention. The hourly
kinetics of intestinal calcium absorption throughout the day
is also of great importance because of the lack of overlap
between the period of uterine deposition of calcium for
shell during the night and the period of dietary calcium
intake during the day. Hens show a specific appetite for
calcium a few hours before the period of calcification, i.e., a
few hours before lights off (Mongin and Sauveur, 1979).
Diet and Ca-particles, when available, are stored in the
crop. Dilatation of the crop elicits an increased acid
secretion (Ruoff and Sewing, 1971; Lee et al., 1988). This
specific appetite for calcium in hens therefore favors the
storage and solubilization of the dietary calcium throughout
the night especially when available as coarse particles,
which partially compensates for the gap in time between
dietary calcium intake and its requirement for shell for-
mation. The timely provision of coarse calcium particles in
this way also limits the hen’s need to mobilize calcium
from the bone reserve and therefore also decreases the
associated elimination of phosphorus (Whitehead, 2004).
The shell contains 60% of carbonate originating from
the blood CO2, which penetrates the uterine glandular cells
by simple diffusion through the plasma membrane (Hodges
and Lörcher, 1967). Carbonic anhydrase 2 (CA2) catalyzes
the reversible hydration of intracellular CO2 to HCO3
.
Bicarbonate is also supplied at a low level from plasma by
the Naþ
/HCO3
cotransporters (SLC4A4, A5, and A10)
(Jonchère et al., 2012; Brionne et al., 2014). The carbonic
anhydrase (CA) present in the uterine tubular gland cells is
crucial for production of bicarbonate which is secreted into
the uterine fluid through the HCO3
/Cl
exchanger
SLC26A9. Another less expressed CA, CA4, is also found
in the uterine cells, its active site being localized in the
extracellular space (Zhu and Sly, 1990). The precipitation
of CaCO3 in the uterine lumen provides Hþ
ions, which are
reabsorbed by the uterine cells. Hþ
ions are exported via
membrane Caþþ
pumps, the vacuolar (Hþ)-ATPase (VAT)
pump, and an Hþ
/Cl
exchanger (Jonchère et al., 2012;
Brionne et al., 2014). These pumps and exchangers control
the pH balance and therefore contribute to maintenance of
acid-base equilibrium in hens. The metabolic acidosis due
to acidification of uterine fluid and plasma during shell
formation is corrected in hens by respiratory hyperventi-
lation and by an increased renal excretion of hydrogen
(Mongin, 1978).
32.4 Hormones involved in calcium
metabolism of laying hens:
vitamin D, parathyroid hormone,
calcitonin, and fibroblast growth
factor-23
Regulation of the extracellular Ca2þ
concentration is
continuously challenged in hens by their large Ca require-
ment for shell formation; however, hens efficiently main-
tain Ca homeostasis by implementing the classical feedback
mechanisms present in all vertebrates. These involve the
intestines, bone, and kidney, and utilize three main calcium
regulating hormones [PTH, calcitonin (CT), and 1,25-
dihydroxy vitamin D3 (1,25(OH)2D3)]. FGF23 is a bone-
derived hormone, which controls phosphorus homeostasis
by suppressing phosphate reabsorption and vitamin D
hormone synthesis in the kidney(Quarles, 2012). This
mechanism has been recently revealed in birds, and the
evidence demonstrating its influence on Ca metabolism are
discussed in this section. These calcium-regulating hor-
mones were initially studied in mice and human, revealing
detailed information for these species. Their roles show
numerous similarities with those in birds even if their
actions and sensitivities are different from mammals
(Dacke et al., 2015). The sex steroid hormones (estrogen
and testosterone) influence Ca metabolism indirectly in
hens at sexual maturity by initiating the formation of
medullary bone and by increasing the appetite for Ca;
however, there is no evidence of their control of Ca ho-
meostasis. Similarly, the uterus is the major contributor to
the elevated need for Ca; however, surprisingly this main
organ of shell formation does not seem to be influenced by
the Ca-regulating hormones even if the ionic transporters
show numerous similarities at the uterine and intestinal
levels (Bar, 2009; Nys and Le Roy, 2018). Other putative
Ca and bone-regulating factors might influence Ca meta-
bolism. Dacke et al. (2015) described the avian-specific
actions of prostaglandins, calcitonin gene-related peptides
(CGRPs), and amylin in pathways that are different from
those in mammals. However, this aspect will not be dis-
cussed further in this review.
32.4.1 Regulation of vitamin D metabolites in
hens
Vitamin D is essential for maintaining egg production and
shell quality in hens. Its regulation in hens has been
reviewed by many authors, including Hurwitz (1989), Nys
(1993), Bar (2008, 2009), Christakos et al. (2014, 2019),
and Nys and Le Roy (2018). One particularity of birds
compared to mammals is the higher biological activity of
vitamin D3 relative to vitamin D2 (ergocalciferol) because
of the lower affinity of avian plasma vitamin D binding
818 PART | V Endocrine theme
8. proteins (DBPs) for vitamin D2 compared to D3 derivatives
(DeLuca et al., 1988). Both vitamin D metabolism and
regulation show large similarities in mammals and birds, but
the magnitude of the fluctuations in hens relative to mam-
mals is considerably larger. Vitamin D3 is partly synthe-
sized from 7-dehydrocholesterol in the skin in response to
UV light, but is mainly provided by diet in commercial hens
(Bar, 2008; Nys, 2017). The minimum daily requirement in
hens was established at 7.5 mg/kg diet (corresponding to a
requirement of about 1 mg/day/hen), according to earlier
studies (Whitehead, 1986; Barroeta et al., 2012). The cur-
rent recommendations are higher for hens producing more
than 330 eggs in a laying year (50 mg/kg diet; 6 mg/bird/day)
(Weber, 2009), which has a positive effect on bone strength
and egg production (Barroeta et al., 2012).
Vitamin D3 is initially hydroxylated in liver microsomes
and mitochondria to form 25-hydroxyvitamin D (25(OH)
D3) (Bar, 2008; Christakos et al., 2014, 2019). This first
hydroxylation is poorly regulated in contrast to the second
hydroxylation step which occurs in the kidney to form the
1,25(OH)2D3 metabolite. Therefore, the plasma levels of
25(OH)D3, which circulate as a complex with vitamin
DBPs, mainly reflect the dietary supply of vitamin D3. Its
plasma level is however not directly proportional to the
dietary supply of vitamin D3 and circulating 25(OH)D3
tends to plateau. Its biological activity is slightly higher in
birds than the nonhydroxylated form, possibly as a conse-
quence of better intestinal absorption.
A longitudinal study describing the changes in plasma
DBPs and 1,25(OH)2D3 throughout embryonic develop-
ment, followed by rearing of pullets (immature hens and
males) until the onset of egg production at week 15e16, is
shown in Figure 32.5 (Nys et al., 1986a). Vitamin
1,25(OH)2D3 is secreted first at embryonic day 13 (Mor-
iuchi and Deluca, 1974), then its blood levels and that of
DBP increase at hatching, possibly as a consequence of
liver maturation. An additional increase occurs at sexual
maturity under the influence of estrogens, as demonstrated
by the increased 1,25(OH)2D3 induced by treatment of
immature pullets with progesterone, testosterone, or estra-
diol (Montecuccoli et al., 1977; Baksi et Kenny, 1977; Nys
et al., 1986a). The interruption of egg production induced
by nutritional deficiencies is accompanied by decreases in
the secretion of sex steroids and reduced concentrations of
DBP and 1,25(OH)2D3. The resumption of egg production
coincides with stimulation of 1,25(OH)2D3 plasma levels
0
100
150
200
250
300
0
100
200
300
400
500
600
700
800
900
0
100
200
300
400
500
600
700
800
100
150
200
250
300
350
DBP ( )
Age (weeks)
0 5 15 20
10 25
Plasma 1,25(OH)2D3 (ng/l)
50
0
Age (weeks)
0 5 15 20
10 25
Osteocalcin (μg/l)
50
Estradiol (μg/l)
mg/l
FIGURE 32.5 Evolution of plasma 1,25(OH)2D3, vitamin D-binding protein, estrogens, and osteocalcin concentrations during the growth of female
(continuous line) and male (dotted line) chickens from birth to age of 25 weeks. The light orange band indicates the period of sexual maturity (Nys, 1993).
Mechanisms and hormonal regulation of shell formation Chapter | 32 819
9. (Nys et al., 1986a, 1986b). Renal production of
24,25(OH)2D3 is seven- to ninefold higher than that of
1,25(OH)2D3 in immature pullets (Montecuccoli et al.,
1977). However, at the onset of laying, hens activate kid-
ney 25-hydroxycholecalciferol 1a-hydroxylase which
elicits a large increase in plasma concentrations of
1,25(OH)2D3 (Figure 32.5) and in intestinal 1,25(OH)2D3,
in contrast to 25(OH)D3 24-hydroxylase (Spanos et al.,
1976; Castillo et al., 1979). Daily eggshell formation co-
incides with slightly higher blood levels of 1,25(OH)2D3.
Its levels are around 100 pmol/L in immature pullets, rising
to more than 200 pmol/L in hens laying shell-less eggs;
however, this doubles in hens laying hard-shell eggs (Nys
et al., 1986a). The stimulation of 1,25(OH)2D3 depends on
two types of regulation. The first is associated with sexual
maturation, while the second results from changes in Ca
metabolism induced by calcium exportation for eggshell
formation. These observations are in agreement with
numerous studies, as reviewed by many authors (Bar, 2008;
Dacke et al., 2015, Nys and Le Roy, 2018). The increased
1,25(OH)2D3 circulating levels in laying hens, compared to
immature pullets, is due to stimulation by estrogens and, at
a lower magnitude, by testosterone (Montecuccoli et al.,
1977; Castillo et al., 1979; Baksi et Kenny, 1977). Estro-
gens act directly on the kidney production of 1,25(OH)2D3
as shown in vitro (Baksi and Kenny, 1977; Tanaka et al.,
1978), but with a lower magnitude than in vivo. Estrogens
might act indirectly through the induced calcium deficiency
(Bar and Hurwitz, 1979) due to the formation of medullary
bone under the combined effect of sex steroids (Bar et al.,
1978; Dacke et al., 2015). However, hens laying eggs with
a soft shell due to artificial premature egg expulsion, and
fed a high-dietary calcium diet, developed hypercalcemia
during the entire laying cycle, while still exhibiting a
relatively high level of plasma 1,25(OH)2D3 (Figure 32.6).
Sex steroids, therefore, can favor a high secretion of
1,25(OH)2D3, even when PTH secretion is abolished and
medullary bone is poorly mobilized. The largest stimulation
in kidney production of 1,25(OH)2D3 results from the hy-
pocalcemia induced by shell formation at sexual maturity
and to a lesser degree during the daily period of shell for-
mation. Hypocalcemia causes increased secretion of PTH,
which substantially increases the production of
1,25(OH)2D3 in vivo (Garabedian et al., 1972) and in vitro
(Trechsel et al., 1979). During the period of eggshell for-
mation, the decrease in plasma ionized calcium occurs in
hens laying hard-shelled eggs, but not in hens laying shell-
less eggs (Figure 32.6). The hypocalcemia increases plasma
PTH (van de Velde et al., 1984a; Singh et al., 1986; Yang
et al., 2013; Kerschnitzki et al., 2014). Plasma hypocalce-
mia and PTH secretion are therefore the predominant
factors stimulating 1,25(OH)2D3 production, as demon-
strated by the threefold increase of 1,25(OH)2D3 induced in
hypocalcemic hens fed a low-calcium diet (1% dietary
calcium), compared with laying hens fed a normal calcium
diet. Additional factors are thought to stimulate in vitro
kidney production of 1,25(OH)2D3, such as prolactin
(Spanos et al., 1979), CT, and growth hormone, but are
probably minor regulators of 1,25(OH)2D3 in vivo in
mature hens (Bar, 2008; Dacke et al., 2015).
More recently, knockout experiments in mice have
revealed a novel circulating factor involved in phosphate
and calcium metabolism. Fibroblast Growth Factor-23
(FGF23) is a bone-derived hormone that suppresses phos-
phate reabsorption and vitamin D hormone synthesis in the
kidney (Quarles, 2012; Erben, 2018). Experimental
approaches in mice have revealed the physiological
importance of FGF23 in inhibiting renal 1a-hydroxylase
(CYP27B1) transcription, which is the key enzyme for
1,25(OH)2D3 synthesis (Shimada et al., 2004). FGF23 de-
creases serum levels of inorganic phosphate by inhibiting
0
20
40
60
80
100
120
140
160
0 5 10 15 20 25
0.6
0.8
1
1.2
1.4
1.6
1.8
200
400
600
800
1000
1200
1400
1600
1800
2000
calcit
e
cucle
EGS
mb.
mam. core
hrs aer ovulaon
4 8 10 12 22
16
Eggshell formaon
0,8
1
1,6
1,8
1,4
1,2
40
0
60
0
10
00
14
00
18
00
16
00
12
00
80
0
40
60
10
0
14
0
16
0
12
0
80
20
Osteocalcin
(μg/l)
Ionized
Ca
(mmol/l)
1,25(OH)
2
D
3
(pmol/l)
shell-less egg
normal eggshell
low dietary Ca
normal
eggshell
FIGURE 32.6 Plasma levels of osteocalcin, ionized calcium, and
1,25(OH)2D3 throughout the ovulatory cycle. Eggshell formation takes
place from 10 to 22 h after ovulation. Normal eggshell corresponds to hens
laying hard-shelled eggs and fed 3.5% dietary calcium. Shell-less eggs
were obtained by premature expulsion of the eggs before shell formation
for a period of 4 days in hens fed 3.5% calcium. The low-dietary calcium
corresponded to hens fed 1% dietary calcium and laying hard-shell eggs.
EGS mb., eggshell membranes; mam. core, mammillary core (Nys, 1993;
Nys and Le Roy, 2018).
820 PART | V Endocrine theme
10. renal phosphate reabsorption and calcitriol production and
is suspected to have a large physiological role in phosphate
homeostasis. Phosphate concentrations are less tightly
regulated than serum ionized calcium levels, but are
maintained in a very limited range thanks to PTH and
active vitamin D. Recent evidence suggests a similar
regulation of phosphate and calcium metabolism by FGF23
in laying hens.
32.4.2 Role of fibroblast growth factor-23 in
regulation in calcium and phosphorus
metabolism
FGF23 (phosphatonin) is a 32 kDa glycoprotein mainly
produced in bone by osteoblasts and osteocytes stimulated
by hyperphosphatemia, as reviewed in Erben (2018). High-
dietary phosphorus stimulates FGF23 production in
humans (Ferrari et al., 2005; Antoniucci et al., 2006). Its
role in the control of phosphate (P) homeostasis was
revealed when excessive FGF23 was discovered to be the
factor responsible for inherited hypophosphatemic rickets
in young children (White et al., 2000). FGF23 belongs to
the family of endocrine FGFs and requires the trans-
membrane co-receptors a- and b-Klotho for high-affinity
binding to the ubiquitously expressed FGF receptors
(FGFR1-4) in target cells (Urakawa et al., 2006; Goetz
et al., 2007). Among the four different FGFRs, FGF
receptor-1c (FGFR1c) is probably the most important
FGFR for FGF23 signaling, at least under physiological
conditions (Urakawa et al., 2006). aKlotho enhances the
binding affinity of FGFR1c to FGF23 by a factor of
approximately 20 (Goetz et al., 2012). In humans, diseases
characterized by excessive blood concentrations of intact
FGF23 lead to renal phosphate wasting and inappropriately
low circulating 1,25(OH)2D3 levels in patients with normal
kidney function (Martin et al., 2012). In the absence of
FGF23 or its co-receptor a-Klotho, the endocrine control of
1a-hydroxylase transcription fails, leading to inappropri-
ately high expression and activity of this enzyme. Hyper-
calcemia, hyperphosphatemia, and impaired bone
mineralization are observed in a-Klotho and FGF23 defi-
cient mice (Yoshida et al., 2002; Shimada et al., 2004). In
addition, FGF23 might stimulate the expression of the 24-
hydroxylase (CYP24A1) which hydrolyses 1,25(OH)2D3 to
the inactive 1,24, 25(OH)2D3 (Liu and Quarles, 2007).
In mammals, FGF23 reduces renal phosphate reab-
sorption by inhibiting the activity of type IIa and type
IIc phosphate transporters, which are responsible for
reabsorption of phosphate from the glomerular filtrate
(Gattineni et al., 2009). FGF23 promotes renal phosphate
excretion by inhibiting cellular phosphate reuptake from the
urine in proximal renal tubules through a cascade involving
the a-Klotho/FGFR1c receptor complex. FGF23 induces
the phosphorylation of the scaffolding protein Naþ
/Hþ
exchange regulatory cofactor (NHERF)-1 which in turn
leads to degradation of the sodium-phosphate cotrans-
porters NaPi-2a and NaPi-2c (Andrukhova et al., 2012). In
addition, in distal convoluted tubules, FGF23 increases
reabsorption of calcium and sodium by increasing the
apical membrane abundance of the epithelial calcium
channel, the transient receptor potential vanilloid-5
(TRPV5) and of the sodium-chloride cotransporter NCC
(Erben, 2018). In chicken, its function on phosphate
homeostasis was initially revealed by the use of antibodies
against FGF23. Immunosuppression of FGF23 greatly
improved phosphate utilization by young chicks, by
inhibiting the stimulation of renal excretion by FGF23
(Bobbeck et al., 2012). Similarly, laying hens immunized to
produce anti-FGF23 antibodies had reduced phosphate
excretion (Ren et al., 2017) and improved eggshell quality
(Ren et al., 2018). In hens, FGF23 expression in the liver is
increased at sexual maturity (Gloux et al., 2019) and during
the period of shell formation in the medullary bone (Hadley
et al., 2016).
In hens and in chicken of different ages, FGF23 mRNA
was expressed at higher levels in liver than other tissues
evaluated, including calvaria, femur, tibia, medullary bone,
brain, spleen, duodenum, jejunum, ileum, heart, and kid-
ney; however, the highest expression of a-Klotho was
found in kidney. It was also expressed in tibia but at a lower
level. High-dietary phosphorus stimulates its expression in
bone but not in liver (Wang et al., 2018).
Synthesis of FGF23 by medullary bone was confirmed
by Gloux et al. (2019). They observed an overexpression of
FGF23 in aged hens compared to young hens, which was
associated with lower plasma levels of 1,25(OH)2D3. In
addition, FGF23 is overexpressed during the period of shell
formation in younger hens when phosphoremia is elevated,
in agreement with Hadley et al. (2016), but this change in
FGF23 production was not observed in older hens. On the
other hand, the ligands of FGF23 at the kidney level,
FGFR2 and FGFR3, do not vary throughout the daily
period of egg formation or with age of hens (Gloux et al.,
2019). It is noteworthy that parathyroid cells in humans
express both aKlotho and FGFRs, but the effect of FGF23
on the parathyroid gland (PTG) remains controversial
(Goetz et al., 2012) and has not been yet explored in hens.
In hens, the recommendation for dietary phosphorus has
been largely decreased compared to 30 years ago because
high levels negatively affect eggshell quality by an un-
known process. FGF23 which is stimulated by high-dietary
phosphorus and reduces the production of 1,25(OH)2D3
might explain this negative impact of phosphorus on shell
quality as suggested by the negative correlation between
shell quality and plasma levels of phosphorus during the
period of shell calcification (Sauveur and Mongin, 1983).
FGF23 might have physiologically functions on human
bone mineralization, as suggested by its powerful inhibitory
Mechanisms and hormonal regulation of shell formation Chapter | 32 821
11. effect on transcription of tissue nonspecific alkaline
phosphatase (TNAP) mRNA in bone cells in a Klotho-
independent manner (Murali et al., 2016). TNAP is
essential for the regulation of bone mineralization by
cleaving the mineralization inhibitor pyrophosphate, which
is secreted by osteoblasts, to prevent premature minerali-
zation of osteoid (Addison et al., 2007). Locally produced
FGF23 may also serve as a physiological inhibitor of bone
mineralization by downregulating TNAP expression. This
possibility has not been yet explored in birds.
32.4.3 Parathyroid hormone and related
peptides
32.4.3.1 Chemistry, secretion, and function
of parathyroid hormone
The principal role of PTH is to regulate blood calcium
concentration and to maintain calcium homeostasis. This
system has been reviewed by many authors in humans and
mice (Jüppner et al., 2000; Potts, 2005; Guerreiro et al.,
2007), and in hens (Hurwitz, 1989b; Dacke, 2000; Dacke
et al., 2015). Parathyroidectomy in birds leads to hypo-
calcemia, tetany, and death (Kenny, 1986), and recipro-
cally, PTH injections into birds increases plasma Ca levels
(Kenny and Dacke, 1974), this effect being larger in laying
birds. Changes in plasma Ca2þ
concentrations are detected
by the CaSR, which is expressed by the PTG chief cells that
also store and secrete PTH (Hurwitz, 1989b). Increases in
plasma Ca2þ
levels lead to increased expression of the
CaSR gene. Its expression in the PTG appears therefore to
be inversely associated with changes in plasma Ca2þ
(Yarden et al., 2000). However, it remains stable
throughout the laying cycle in hens (Gloux et al., 2020a).
CaSR is also expressed in bone and kidney (Courbebaisse
and Soubervielle, 2011), and contributes to the physiolog-
ical responses of these organs to maintain Ca homeostasis.
Two (chicken) to four (Japanese quail) PTGs are present in
birds, near the thyroids (Dacke et al., 2015). Sequences of
mammalian forms of PTH and chicken PTH share struc-
tural homology and overlap in function (signal using the
same G protein-linked receptor) (Potts, 2005). All
mammalian PTH molecules consist of a single chain
polypeptide with 84 amino acids and a molecular weight of
approximately 9400 Da (Potts, 2005). The amino acid
sequence deduced from the DNA sequence showed that
chicken prepro-PTH mRNA encoded a 119 amino acid
precursor and an 88 amino acid hormone (Lim et al., 1991).
The sequence of chicken PTH shows significant differences
in comparison with its mammalian homologs; for example,
avian PTH contains two deletions in the hydrophobic
middle portion of the sequence and an additional 22 amino
acids near the C-terminus, which replaces the stretch of
nine amino acids, residues 62 to 70, in the mammalian
hormones (Potts, 2005). The amino terminal region of PTH
is the minimum sequence necessary and sufficient for
regulation of mineral ion homeostasis and shows high-
sequence conservation among all vertebrate species
(Jüppner et al., 2000). This 1e34 sequence is also present
in the PTH-related peptide (PTHrP) and in a third PTH-like
peptide (PTH-L) identified in chicken (Guerreiro et al.,
2007; Pinheiro et al., 2010). These three peptides share the
highly conserved N-terminal region, which controls Ca
homeostasis and skeletal development. PTHrP and PTH-L
shows a widespread and complex tissue distribution in
vertebrates, which suggests their involvement in paracrine
regulation (Pinheiro et al., 2010). Their role in non-
mammalian vertebrates has not been fully explored.
The actions of PTH on mineral ion homeostasis in bone
and kidney is mediated by a single receptor, the PTH-
PTHrp receptor (PTHR1), which belongs to a distinct
family of G protein-coupled receptors, as revealed by
cloning in several species (Juppner et al., 2000; Potts
2005). This family of B receptors have a long amino ter-
minal extracellular domain that is critical for binding pep-
tide ligands such as PTH. The cloning of the receptor and
studies of structureeactivity relations with the PTH ligand
and receptor has allowed the cellular biology of PTH action
in various tissues to be explored (Jüppner et al., 2000;
Potts, 2005; Courbebaisse and Soubervielle, 2011).
32.4.3.2 Regulation by parathyroid hormone
of Calcium metabolism
The primary physiological role of PTH and its related
proteins is to maintain in the short term the circulatory
levels of ionic calcium. Hypocalcemia is detected by the
PTGs and induces PTH secretion (Garabedian et al., 1972).
When calcium is needed (calcium-deficient diet, vitamin D
insufficiency, or eggshell formation), calcium is rapidly
mobilized from bone, in particular medullary bone in laying
birds, in response to increased PTH secretion. In addition,
PTH stimulates calcium absorption over the longer term by
increasing the synthesis of 1,25(OH)2D in the kidney
though activation of the 25-hydroxycholecalciferol-1e
hydroxylase (Fraser and Kodicek, 1973). PTH also pro-
motes phosphate excretion by blocking its reabsorption,
leading to excretion of the phosphate excess liberated by
bone resorption and reduces the excretion of urinary cal-
cium by increasing renal calcium reabsorption at distal
tubular sites in the kidney.
In bone, PTH has multiple catabolic and anabolic ef-
fects that affect the skeleton (Teitelbaum, 2000; Parra-
Torres et al., 2013; Dacke et al., 2015). A number of cell
lines of osteoblasts and stromal cells, utilizing specialized
tissue culture systems, have evolved to study the interaction
between different cell types and their role in bone formation
and bone resorption (Potts, 2005). Through its abundant
822 PART | V Endocrine theme
12. receptors on osteoblasts, in contrast to osteoclasts, PTH has
a variety of actions that are directly involved in promoting
bone formation; however, physiologically, its most impor-
tant role is to stimulate osteoclast differentiation and
development and ultimately increase bone resorption. It has
been shown in vitro that the maturation of macrophages
into osteoclasts requires the presence of marrow stromal
cells or their osteoblast progeny (Teitelbaum, 2000).
Osteoclast differentiation is indirectly induced by osteo-
blasts that express the membrane-bound receptor for acti-
vation of nuclear factor kappa B (NF-kB) (RANK) ligand
(RANKL) and macrophage colony-stimulating factor (M-
CSF) (Teitelbaum, 2000). M-CSF binds to its receptor, c-
Fms, on early osteoclast precursors, and provides signals
required for their survival and proliferation. A direct con-
tact between RANKL-expressing osteoblasts and RANK-
possessing osteoclasts, and their progenitors, is essential
for osteoclastogenesis during bone development (Yasuda
et al., 1998). RANKL is also expressed in osteocytes at
levels several-fold higher than in osteoblasts; therefore,
osteocytes also control RANK-expressing osteoclasts dur-
ing bone remodeling (Nakashima et al., 2012). This process
is also regulated by a secreted decoy receptor of RANKL,
osteoprotegerin (OPG), which functions as a paracrine in-
hibitor of osteoclast formation by competing with RANK
for RANKL (Yasuda et al., 1998). Studies with avian
systems confirm the critical role of RANK, RANKL, and
OPG in birds (Dacke et al., 2015). Human recombinant
RANKL has been shown to stimulate the resorptive activity
of osteoclasts isolated from embryonic chick tibia (Boissy
et al., 2001) and from bone marrow of Muscovy ducks (Gu
et al., 2009). In hens, the hypercalcemic action of PTH is
rapid (30 min), firstly by inhibition of plasma Ca2þ
clearance, followed by altering cell spread area in avian
osteoclasts and by inducing the osteoclasts to form ruffled
borders (Dacke et al., 2015). This effect was observed
in vitro in osteoclasts located within hen medullary bone
(Sugiyama and Kusuhara, 1994). Mechanisms of bone
resorption by osteoclasts are similar in mammals and birds;
a proton pump-ATPase and an Naþ
, Kþ
-ATPase function
in the ruffled border, and a CA and a Ca2þ
-ATPase are
located on the osteoclast plasma membrane. In laying hens,
circulating PTH is implicated in the regulation of bone
resorption, as evidenced by higher levels of circulating
PTH during the period of shell formation, as demonstrated
by a cytochemical bioassay (van de Velde et al., 1984a;
Singh et al., 1986). This bioassay reflects activity of the
1e34 sequence, whatever its origin (PTH, PTHrP, or PTH-
L), but was not observed in a recent study (Ren et al.,
2019). However, an increased expression of PTH mRNA
was also observed in the PTG during this active phase of
eggshell formation (Gloux et al., 2020a). More detailed
information on the role of PTH in other tissues including
kidney, intestine, uterus, and smooth muscle have been
reviewed by Dacke et al. (2015).
32.4.4 Calcitonin and calcitonin gene-related
peptides
CT is a 32-amino acid polypeptide hormone secreted by the
parafollicular cells (C-cells) of the thyroid gland in mam-
mals and by the ultimobranchial tissue in avian and other
nonmammalian species (Felsenfeld and Barton, 2015;
Dacke et al., 2015; Xie et al., 2020). In humans, CT is
released when plasma Ca2þ
levels increase, and protects
against the development of hypercalcemia. The secretion of
CT is modified by the CaSR, as observed for PTH, but,
while activation of the CaSR suppresses PTH secretion, it
stimulates CT secretion. Both hormones also act on bone
and kidney through receptors of the class II subclass of G-
protein-coupled receptors. CTR expression is observed in
the kidney, mature osteoclasts, and other tissues, and it
interacts with receptor modifying proteins to form an active
complex (Xie et al., 2020). CT administration decreases the
magnitude of hypercalcemia during calcium loading in
humans (Felsenfeld and Barton, 2015); however, birds are
refractory to CT administration (Dacke et al., 2015). This
hypocalcemic effect in mammals is attributed to inhibition
of bone resorption by reducing osteoclast activity and to
suppression of calcium release from the bone. In addition,
CT increases renal production of 1,25D in the convoluted
proximal tubule (Felsenfeld and Barton, 2015). Recipro-
cally, CT gene transcription is suppressed by 1,25(0H)2D3.
There is therefore evidence that CT administration in hu-
man is effective; however, since its discovery more than 50
years ago, there is no evidence that any deficiency or excess
of CT observed in various human pathologies results in
skeletal abnormalities. Calcium metabolism and bone
mineral density are not affected in patients with medullary
thyroid carcinoma with a chronically increased level of
endogenous CT, or in thyroidectomized individuals with
undetectable circulating CT (Xie et al., 2020). A precise
role for CT remains elusive in humans and its function in
nonmammalian vertebrates is even less understood (Fel-
senfeld and Barton., 2015). However, others still consider
that under calcium stress conditions, CT might play a vital
role in protecting the skeleton (Xie et al., 2020) because CT
suppresses the bone disruption process in CT-deficient
mice, as compared to wild-type mice. Dacke et al. (2015)
reviewed some evidence in birds showing that in vitro CT
can affect osteoclasts from Ca-deficient chicks. Cultured
medullary bone osteoclasts from these birds respond to CT
administration by a reduction in cell spread area, and by
inhibiting the bone-resorptive activity or by suppressing
ruffled borders. However, there is also experimental evi-
dence that might explain why CT is not efficient in vivo to
Mechanisms and hormonal regulation of shell formation Chapter | 32 823
13. correct hypercalcemia in birds. Dacke et al. (2015)
concluded that CT plays a minor role in regulating Ca
metabolism in birds. The difference between effects of CT
on bone cells in vitro and in vivo in human, as well as the
large variation in response between different species, brings
into question the role of CT in Ca metabolism, particularly
in birds.
Additional CGRPs, including CGRPs (alpha CGRP and
beta CGRP), amylin, adrenomedullin, and intermedin, also
contribute to bone regulation. In vitro and in vivo studies
concluded that there were no inhibitory effects on osteo-
clasts and bone resorption and observed positive effects on
osteoblasts and bone formation in mice and human. More
information on their putative functions in mammals can be
found in recent reviews (Naot et al., 2019; Xie et al., 2020).
In birds, the roles of CGRP and amylin have been described
by Dacke et al. (2015). CGRP is a 37 amino acid neuro-
peptide derived from the same gene as CT (amylin super-
family) and is expressed in many tissues of the central
nervous system, including neurons innervating bone.
CGRP might interact with osteoclastic CT receptors sug-
gesting a paracrine role in modulation of bone turnover.
A few studies show that CGRP in vivo elicits hypercalce-
mic and hypophosphatemic responses in chicks (Dacke
et al., 2015), but very little experimental work has been
carried out in birds and CGRP involvement in regulation of
Ca metabolism remains hypothetical.
32.5 Intestinal absorption of calcium
32.5.1 Mechanisms of intestinal calcium
absorption
Body calcium is ultimately derived from the diet, and
intestinal absorption is therefore critical for calcium bal-
ance. Intestinal calcium uptake occurs in mammals through
two defined mechanisms, a nonsaturable paracellular
transport pathway, which results from passive diffusion and
an active transcellular pathway (Courbebaisse and Sou-
bervielle, 2011; Ghishan and Kiela, 2012; Christakos et al.,
2014, 2019). High-dietary intake of calcium enables
absorption by passive transport, but in humans, the dietary
supply of calcium content is in the lowenormal range, and
the active transcellular pathway is required. This process
occurs in the proximal part of the intestine (duodenum and
jejunum) which shows a greater capability for calcium
absorption than the distal segment. However, net calcium
absorption might still be predominant in the ileal segment
by passive absorption (up to 80%) due to the long-transit
duration (Wasserman, 2004). Similarly, in birds, the
intestinal absorption of calcium is controlled by these two
distinct transport processes (Hurwitz, 1989a; Bar, 2009;
Nys and Le Roy, 2018). In chicks and hens, the proximal
intestine shows a high efficiency to absorb calcium, as
demonstrated by direct measurement of Ca absorption us-
ing Yttrium-91 or Ca-45 (Hurwitz and Bar, 1966;
Bar, 2009). The duodenum has a higher capacity for Ca
absorption than the jejunum, but the transit duration is
longer in the jejunum due to its greater length. In birds,
most of the calcium is therefore absorbed before it reaches
the lower ileum, as a result of the higher efficiency of the
proximal intestine in absorbing Ca2þ
, and of the lower ileal
electrochemical potential difference (Bar, 2009). The high
magnitude of calcium absorption (threefold greater in
mature hens than in immature pullets), the huge increase in
intestinal calbindin 28kD concentration in laying hens, the
high correlation of calbindin with the capacity to absorb
calcium, and the stimulation by vitamin D metabolites of
key proteins involved in the transcellular pathway (Bar,
2009; Nys and Le Roy 2018), all underline the importance
of the active cellular process in calcium absorption in hens.
However, the paracellular pathway, which is stimulated by
a favorable gradient of Ca2þ
concentration between the
intestinal and plasma compartments, might contribute to
the large daily increase in Ca retention observed during the
eggshell formation period (Hurwitz and Bar, 1965), as this
is consistent with the higher levels of solubilized calcium in
the intestinal lumen (Guinotte et al., 1995). The larger
concentration of solubilized Ca in the intestinal content of
the proximal intestine results firstly from the large intake of
dietary calcium due to a specific appetite for calcium a few
hours before shell formation (Mongin and Sauveur, 1974;
Wilkinson et al., 2011), and secondly from the stimulation
of acid secretion induced by dilatation of the crop a few
hours before nightfall (Mongin, 1976). Inhibition of acid
secretion by omeprazole (inhibitor of Hþ
, Naþ
-ATPase)
reduces net calcium intestinal retention by 20%, confirming
the importance of the calcic gradient (Guinotte et al., 1995).
The use of dietary calcium with a large particle size
reinforces the supply of calcium for a longer period in
the intestinal content, and reduced the gap between the
intestinal supply of Ca and its uterine exportation for
the eggshell formation, which mainly take place during the
night. The use of large particles of dietary Ca is therefore
very frequent in practical conditions and is well known for
more than 50 years to improve shell strength (Nys, 2017).
In conclusion, both passive and active processes in hens
contribute to optimize the Ca retention needed for shell
formation.
Both mechanisms of Ca absorption, the transcellular
active transport and the paracellular nonsaturable passive
pathway, are regulated by 1,25 dihydroxyvitamin D3 and
have been biochemically characterized in mammals and
birds (Christakos et al., 2014, 2019; Bar, 2008; Nys and Le
Roy, 2018). The active transcellular calcium absorption
involves the transfer of calcium across the luminal brush
border membrane, through the cell interior and its extru-
sion from the basolateral membrane. In mammals
824 PART | V Endocrine theme
14. (Wasserman, 2004; Christakos et al., 2014, 2019), calcium
entry occurs through the epithelial calcium-selective
channel TRPV6 (transient receptor potential cation chan-
nel subfamily V member 6). It belongs to a super-family of
cation channels (30 different TRP subunit genes proteins
in six subfamilies in mammals). TRP channels operate
either as primary detectors of chemical and physical
stimuli, as secondary transducers of ionotropic or metab-
otropic receptors, or as ion transport channels (Zheng,
2013; Alaimo and Rubert, 2019). Functional TRP chan-
nels, thought to be composed as homo- or heterotetramers,
are opened or closed by conformational changes in the
channel protein (Holzer, 2011). TRPV5 and TRPV6 are
the most Ca2þ
-selective members of the TRP ion channel
family and play an important role in intestinal or kidney
Ca2þ
transfer in mammals (Holzer, 2011). TRPV6 is
however predominantly expressed in intestinal epithelial
cells while TRPV5 is more present in the kidney (Nijen-
huis et al., 2005; Ko et al., 2009). TRPV6 has been iden-
tified by some authors (Yang et al., 2011; Jonchere et al.,
2010, 2012; Huber et al., 2015; Li et al., 2018) in the in-
testine of the laying hen, but was not detected by others
(Proszkowiec-Weglarz and Angel, 2013; Juanchich et al.,
2018; Proszkowiec-Weglarz et al., 2019; Gloux et al.,
2019). An immunoreactive TRPV6 protein was however
revealed in the chicken small intestine by Western blotting
(Huber et al., 2015). Additional TRPVs (TRPV2 but not
TRPV5), TRPM7, and TRPC1 genes are expressed in the
various part of the intestine of laying hens (Li et al., 2018;
Gloux et al., 2019). A summary of ionic transporters in hen
intestine is presented in Figure 32.7, and a list of putative
ionic transporters in the intestine are reported in
Table 32.1.
Following its entry, calcium accumulates within several
minutes in the subapical brush border (Fullmer and Was-
serman, 1987; Chandra et al., 1990). Cytosolic calbindin
(9K in mammals, 28 kD in chicken) binds calcium with
high affinity and facilitates the transcytosolic diffusion of
calcium from the subapical zone to the basal membrane as
reviewed by numerous authors (Wasserman, 2004; Ghishan
and Kiela, 2012; Christakos et al., 2014, 2019; Hurwitz and
Bar, 1989; Bar, 2009; Nys and Le Roy, 2018). Calbindins
are present at high levels in all tissues that transfer large
amount of Ca, including intestine and uterus (Wasserman
and Taylor, 1966; Corradino et al., 1968). Its presence is
considered to be a biomarker for regulated Ca transfer,
since calbindin concentration is well correlated with the
capacity of a tissue to transfer Ca. Ca may also be
sequestered by the endoplasmic reticulum (ER) to prevent
increased levels of intracellular calcium in the enterocyte
(Christakos et al., 2014). Genes of the ITPR family, which
are involved in Ca2þ
extrusion from the ER, are expressed
and modulated by sexual maturity in laying hens (Jonchere
et al., 2012; Gloux et al., 2019). The intestinal plasma
plasma
T
intesne lumen
VDR
N
Ca2+
Ca2+
Na+
2H+
Ca2+
apical
basal
EnRe
Ca2+
2H+
Ca2+
ATP2B1/2 ATP2A2/3
ITPR1/2/3
CALB1 Calcium
TRPV 6, 2, 4 TRPC1
SLC8A1
ATP2B1
TRPM7 CACNA1D 1E 1H
Paracellular
pathway
1,25(OH)2D3 VDR
ATP2A1, 2, 3
ITPR1, 2, 3
Tight juncon
OCLN
Jam2
CLD
2, 12
TJP1
TJP3
TJP2
TJP1
TJP3
TJP2
TJP1
TJP3
TJP2
FIGURE 32.7 General model describing intestinal ion transporters acting in the enterocytes of the laying hen. All symbols are defined in the legend part
(right). EnRe, endoplasmic reticulum; N, nucleus. Data are compiled from Jonchére et al. (2012); Brionne et al. (2014); Gloux et al. (2019, 2020).
Mechanisms and hormonal regulation of shell formation Chapter | 32 825
15. TABLE 32.1 Major proteins involved in ionic transfer at intestinal and uterine level: description, gene expression,
and presence of vitamin D response element and estrogen response element.
Gene
symbol Name
Transfer
type
Cell
location
Tissue expression
Presence of
Gene response
element
Uterus Duodenum VDR EREF
TRPV2, 4,
6
Transient receptor potential
cation channel subfamily V
member2, 4, 6
Ca2þ
channel
PM Y N ? ?
CALB1 Calbindin 28 K Ca2þ
intracellular
transporter
IC Y Y Y Y
Otop2 Otopetrin Ca2þ
intracellular
transporter?
IC Y ? ?
ATP2A2 Endoplasmic reticulum cal-
cium ATPase 2
Ca2þ
ATPase ER Y N N Y
ATP2A3 Endoplasmic reticulum cal-
cium ATPase 3
Ca2þ
ATPase ER Y Y Y Y
ITPR1 IP3 receptor1 Ca2þ
channel
ER Y Y Y N
ITPR2 IP3 receptor2 Ca2þ
channel
ER Y N Y Y
ITPR3 IP3 receptor3 Ca2þ
channel
ER Y Y Y Y
RYR1 Ryanodine receptor 1 Ca2þ
channel
ER Y N Y Y
ATP2B1
PMCA1
Plasma membrane calcium-
transporting ATPase 1
Ca2þ
/Hþ
exchanger
PM Y Y Y Y
ATP2B2
PMCA2
Plasma membrane calcium-
transporting ATPase 2
Ca2þ
/Hþ
exchanger
PM Y Y Y Y
ATP2B4 Plasma membrane calcium-
transporting ATPase 4
(PMCA4)
Ca2þ
/Hþ
exchanger
PM Y Y Y Y
SLC8A1 Sodium/calcium exchanger 1 Naþ
/Ca2þ
exchanger
PM Y Y Y Y
SLC8A3 Sodium/calcium exchanger 3 Naþ
/Ca2þ
exchanger
PM Y Y N N
CACNA
1D, 1E,
1H,
Voltage-dependent L-type cal-
cium channel subunit alpha-
1D, -1E, -1H
CA2 Carbonic anhydrase 2 Catalyze
HCO3
formation
PM Y N Y Y
CA4 Carbonic anhydrase 4 Catalyze
HCO3
formation
PM Y Y N Y
CA7 Carbonic anhydrase 7 Catalyze
HCO3
formation
PM Y Y Y Y
CA9 Carbonic anhydrase 9 Catalyze
HCO3
formation
PM Y N Y Y
826 PART | V Endocrine theme
16. TABLE 32.1 Major proteins involved in ionic transfer at intestinal and uterine level: description, gene expression,
and presence of vitamin D response element and estrogen response element.dcont’d
Gene
symbol Name
Transfer
type
Cell
location
Tissue expression
Presence of
Gene response
element
Uterus Duodenum VDR EREF
SLC26A9 Solute carrier family 26 mem-
ber 9
HCO3
/Cl
exchanger
PM Y Y Y N
SLC4A4 Solute carrier family 4 mem-
ber 4
Naþ
/HCO3
cotransporter
PM Y N Y Y
SLC4A5 Solute carrier family 4 mem-
ber 5
Naþ
/HCO3
cotransporter
PM Y Y N N
SLC4A7 Solute carrier family 4 mem-
ber 7
Naþ
/HCO3
cotransporter
PM Y Y N N
SLC4A10 Solute carrier family 4 mem-
ber 10
Naþ
/HCO3
cotransporter
PM Y N Y Y
SLC4A9 Solute carrier family 4 mem-
ber 9
HCO3
/Cl
exchanger
PM Y Y N N
SCNN1A Amiloride-sensitive sodium
channel subunit a
Naþ
channel PM Y Y N N
SCNN1B Amiloride-sensitive sodium
channel subunit b
Naþ
channel PM Y Y N N
SCNN1G Amiloride-sensitive sodium
channel subunit g
Naþ
channel PM Y Y N N
ATP1A1 Sodium/potassium-transporting
ATPase subunit a-1
Naþ
/Kþ
exchanger
PM Y N N N
ATP1B1 Sodium/potassium-transporting
ATPase subunit b-1
Naþ
/Kþ
exchanger
PM Y N N N
ATP6V1B2 Vacuolar H ATPase B subunit
osteoclast isozyme
Hþ
pump Organelles
and PM
Y N N N
ATP6V1C2 Vacuolar H ATPase B subunit
osteoclast isozyme
Hþ
pump Organelles
and PM
Y Y N N
Annexin-1 Vesicular calcium channels Calcium
entry in
vesicles
Vesicles Y N ? ?
Annexin-2 Vesicular calcium channels Calcium
entry in
vesicles
Vesicles Y Y ? ?
Annexin-8 Vesicular calcium channels Calcium
entry in
vesicles
Vesicles Y N ? ?
Claudin 2,
10, 12
Paracellular cation channel
Tight junction permeability
Paracellular
pathway
Membrane
protein
Y Y ? ?
Jam Junctional adhesion molecule Paracellular
pathway
Actin
cytoskeleton.
Y Y ? ?
TJP 1, 2, 3
ZO 1, 2, 3
Tight junction proteins 1, 2, 3
Zonula occludens 1, 2, 3
Paracellular
pathway
Membrane
protein
Y Y ? ?
OCLN Occludin Paracellular
pathway
Y Y ? ?
ER, endoplasmic reticulum; PM, plasma membrane. References can be found in the text.
Mechanisms and hormonal regulation of shell formation Chapter | 32 827
17. membrane ATPase (ATP2B1 also called PMCA1b), along
with the Naþ
/Ca2þ
exchangers (SLC8A1 or NCX1), per-
forms the final step in transcellular Ca2 absorption,
extruding Ca2þ
from the cell interior to the interstitial space
at the basolateral membrane (Stafford et al., 2017). PMCA1
(ATP2B1) is the only isoform present in enterocytes
throughout the human or mouse intestine and its expression
is higher proximally in the duodenum than in the jejunum
or ileum (Alexander et al., 2015); expression levels are
positively correlated with both intestinal Ca2þ
absorption
and bone mineral density in mice (Replogle et al., 2014).
PMCA1b is also the predominant isomer expressed in the
chicken intestine (Melancon and DeLuca, 1970; Bar, 2009;
Jonchere et al., 2012; Brionne et al., 2014).
The Naþ
/Ca2þ
exchange mechanism (NCX, SLC8A) is
the alternative transporting system involved in the “uphill”
extrusion of Ca2þ
across the basolateral membrane of the
epithelial cell, toward the plasma (Bar, 2009; Liao et al.,
2019). The NCX family contains three separate gene
products exhibiting differential expression; NCX1
(SLC8A1) is mainly expressed in all organs of the digestive
system, while NCX2 and NCX3 are expressed in the ner-
vous system and skeletal muscle (Liao et al., 2019). Naþ
/
Ca2þ
exchange activity is increased in response to calcium
deficiency in chick enterocytes (Centeno et al., 2004). In
laying hens, both NCX1 (SLC8A1) and NCX3 (SLC8A3)
are expressed in the duodenum at a level quite similar to
that of the uterus (Jonchere et al., 2012). The relative
expression of both genes cannot be compared in this tran-
scriptomic approach, but by analogy with mammals, it is
likely that NCX1 predominates, and that PCMA is more
active than NCX1 to extrude Ca2þ
.
The paracellular pathway occurs between adjacent
enterocytes (Figure 32.7). The most apical region of the
intercellular junction is the tight junction (TJ). The barrier
function and the permeability characteristics of epithelial
cell sheets covering different organs are defined by the
properties of the TJ and their paracellular channels (Gunzel
and Yu, 2013; Alexander et al., 2014). TJs are predomi-
nantly formed by claudins, a family of four-transmembrane
proteins with 27 members in human and mouse (Van Itallie
and Anderson, 2014; Zeisel et al., 2019). The physiological
properties of TJs allow selective transport of solutes and
water between compartments, which mainly depends on the
specific subtypes of claudins that they contain. These
membrane proteins function as paracellular cation channels.
Overexpression of claudin 2 (CLDN2) or CLDN12 en-
hances Ca transfer through intestinal epithelial cells in vitro
(Fujita et al., 2008). TJs contain numerous additional pro-
teins, among them transmembrane proteins including the
junctional adhesion molecule (JAM), occludin (OCLN),
and tricellulin. In addition, some cytoplasmic plaque pro-
teins (Zonula occludens, ZO-1, 2, and 3, also called TJ
proteins 1, 2, and 3) are framework-forming proteins that
connect transmembrane proteins with the actin cytoskeleton
(Van Itallie and Anderson, 2014; Zihni et al., 2016; Zeisel
et al., 2019). In laying hens, expression of CLDN1, 2, 10,
and 12, of TJP 1, 2, and 3, of OCLN, and of JAM2 are
observed in duodenum, jejunum, and ileum (Gloux et al.,
2019, 2020a). Only expression of CLDN2 and 10, OCLN,
and JAM2 are affected by age or intestinal location.
Expression of CLDN2 and TJP-1, 2, 3 (ZO1, 2, 3) is higher
in intestine of mature hens compared to that of immature
pullets (Gloux et al., 2019). Expression of CLDN2 and
TJP3 is also slightly increased during the period of shell
calcification (Gloux et al., 2020). Expression of both these
genes and of OCLN is decreased in aged hens compared to
young ones (Gloux et al., 2020). These observations
confirm the relative importance of the transcellular to the
paracellular Ca uptake pathway in laying hens.
32.5.2 Regulation of Calcium absorption in
laying hens
Vitamin D and its metabolites, mainly 1,25(OH)2D3, are
the key factors regulating intestinal Ca2þ
absorption in
mammals and birds (Bouillon et al., 2003; Wasserman,
2004; Bouillon and Suda, 2014; Christakos et al., 2014,
2019; Diaz de Barbosa et al., 2015; Hurwitz, 1989a; Bar,
2008; Nys and Le Roy, 2018). In hens, sexual maturity and
the egg production period coincide with a large increase in
renal production and plasma 1,25(OH)2D3. This large
increase in levels of 1,25(OH)2D3 is responsible for the
large stimulation in net absorption of calcium (Bar et.,
1978; Hurwitz, 1989a: Nys, 1993; Bar, 2008; Nys and Le
Roy, 2018; Gloux et al., 2019). The active form of vitamin
D binds to the intracellular receptor vitamin D receptor
(VDR), which also interacts with retinoic X receptor
(RXR), to form a VDR-RXR complex for binding with a
specific vitamin D receptor element (VDRE) sequence of
the targeted genes in the gene-promoter region; this stim-
ulates the synthesis of mRNAs coding for several proteins
(Schräder et 1993; Dong et al., 2010; Bar, 2008). The
coupled 1,25(OH)2D3 þ VDR has been purified from the
chicken intestine (Pike and Haussler, 1979) and is
expressed in all intestinal segments (Gloux et al., 2019,
2020a), although less expressed in aged hens (Gloux et al.,
2020b). This stimulates the permeation of calcium ions
across the apical surface of enterocytes, as well as its
cytosolic transport and extrusion from the cell (Wasserman
et al., 1992). VDREs were identified in the promoters of
genes encoding TRPV (Weber et al., 2001), calbindins
(Christakos et al., 2014; Nys and Le Roy, 2018), and
PMCAs (Glendenning et al., 2000).
TRPV6 expression is stimulated by low-dietary calcium
and by 1,25(OH)2D3 in mammals (Nijenhuis et al., 2005).
TRPV6 is expressed at a lower level in the duodenum than
in uterus (Jonchère et al., 2012). However, more recent
828 PART | V Endocrine theme
18. studies question the role of TRPV6 as they were unable to
find any expression in the intestine (Gloux et al., 2019,
2020). However, changes in expression of other calcium
TRP channels are observed in hens: TRPM7 and TRPC1
expression increase with age (Gloux et al., 2019), that of
TRPV2 varies with intestinal localization and in response
to Ca diet (Gloux et al., 2020), and expression of TRPM7
and TRPV2 is down-regulated in old hens compared to
young ones (Gloux et al., 2020b); these observations sug-
gest a possible involvement of these additional Ca channels
in enterocyte Ca entry in hens.
Calbindins were intensively studied for their depen-
dence on 1,25-(OH)2D3 (Wasserman et al., 1992; Wasser-
man, 2004; Christakos et al., 2014, 2019; Bar, 2009). In
chickens, calbindin 28kD has been extensively investigated
because of its high levels observed in epithelia transporting
large amounts of calcium. The epithelial capacity to transfer
calcium is highly correlated with calbindin content (Was-
serman et al., 1966; Bar and Hurwitz, 1979). In vitamin D
deficient chicks, CALB1 mRNA is barely detectable in the
intestine (Theofan et al., 1986; Mayel-Afshar et al., 1988)
and is increased approximately 10-fold after injection of
1,25(OH)2D3. Calcium is rapidly (5 to 20 min) transferred
from the intestinal lumen to the epithelium through the
apical surface of enterocytes and accumulates at the sub-
jacent apical zone when vitamin D is deficient. When
supplied with vitamin D, calcium is transferred from the
subapical region to the basal zone of the cells via calbindin
(Chandra et al., 1990). Duodenal calbindin levels are
correlated with changes in 1,25(OH)2D3 plasma levels,
whether induced by restrictions of dietary Ca2þ
or by
exogenous supply of vitamin D derivatives in young chicks
(Wasserman and Taylor, 1966; Bar et al., 1990), or when
egg production is induced or interrupted (Bar et al., 1978;
Nys et al., 1992a; Sugiyama et al., 2007). In chickens,
increased concentrations of plasma 1,25(OH)2D3, increases
in intestinal absorption of calcium and increases in con-
centration of intestinal CALB1 and its mRNA are all
observed to occur at two time points: at the onset of laying
after the hen becomes sexually mature, and at the time of
the formation of the first eggshell (Bar et al., 1978; Nys
et al., 1992a; Striem and Bar, 1991). When immature birds
are not deficient in vitamin D, administration of estrogen
and testosterone stimulates the synthesis of intestinal cal-
bindin (Nys et al., 1992a; Striem and Bar, 1991). However,
the largest increase in calbindin levels is associated with the
onset of egg and shell formation. Induction of the pro-
duction of shell-less eggs by premature expulsion of the
egg for a few days decreases the intestinal level of calbindin
by 50%, while resumption of shell formation stimulates
levels of calbindin protein and its mRNA abundance (Nys
et al., 1992a). The decreased calbindin level and mRNA
expression does not occur in the few hours following egg
expulsion but after a few days in the intestine in contrast to
uterus (Figure 32.8). Arrest of egg production in molted
hens also reduces the intestinal concentration of calbindin
(Bar et al., 1992). These changes in calbindin levels are
synchronized with those of plasma 1,25(OH)2D3. However,
hourly changes in plasma 1,25(OH)2D3 observed during
the daily cycle of egg formation, or alterations induced by
suppression of shell formation by egg expulsion, have no
influence on mRNA expression of intestinal calbindin nor
on the concentration of the protein (Nys et al., 1992a;
Gloux et al., 2020a).
Extrusion of calcium from the enterocyte through the
PMCA1 is also vitamin D dependent. PMCA1 (ATP2B1)
expression is stimulated by the metabolite 1,25-(OH)2D3 in
mice and chickens (Lee et al., 2015) or by factors influ-
encing its metabolism in chicks (Wasserman et al., 1992).
The PMCA pump (ATP2B1) is expressed at a higher level
in the duodenum compared to the uterus during the active
phase of calcium secretion (Jonchère et al., 2012). In hens,
ATPase plasma membrane Ca2þ
transporting 1 (ATP2B1)
and ATPase plasma membrane Ca2þ
transporting 2
(ATP2B2) are expressed in all intestinal segments at
different ages; however, only ATP2B2 expression is greatly
enhanced after sexual maturity in the duodenum, jejunum,
and ileum (Gloux et al., 2019). PMCA1 (ATP2B1) was
slightly increased during the daily period of shell formation
in young hens, but not in older birds (Gloux et al., 2020a,
2020b). However, the Ca2þ
-ATPase activity when
measured globally was similar in the duodenum of hens
calcifying an egg compared to hens laying shell-less eggs
0
20
40
60
80
100
120
0
50
100
150
200
250
Hours aer egg
expulsion
Before egg
expulsion
Duodenal
calbindin
Uterine
calbindin
Protein (μg/mg)
mRNA (/% inial value)
12h p.o. 1h 3h 6h
FIGURE 32.8 Level of expression and concentration of duodenal and
uterine calbindin in hens during the initial phase of shell mineralization of
the shell and 1, 5, and 6 h after experimental expulsion of the egg to
suppress the shell formation Nys et al., 1992a.
Mechanisms and hormonal regulation of shell formation Chapter | 32 829
19. (Nys and de Laage, 1984). The role of sex steroids in the
regulation of duodenal PMCA1 is confirmed by stimulation
of its expression by estrogens, and by the observation that
ovariectomized rats and mice display reduced PMCA1
mRNA (Dong et al., 2014; Van Cromphaut et al., 2003).
Moreover, an estrogen response element (EREF) is present
in the ATP2B1-2 gene, in addition to a VDRE (Nys and Le
Roy, 2018). No information is available on the regulation in
birds of intestinal Naþ
/Ca2þ
exchangers (NCX1).
It has been suggested that vitamin D might also stim-
ulate paracellular calcium absorption, as observed in
mammals (Wasserman, 2004; Christakos et al., 2014). In
hens, the paracellular pathway is of importance due to the
large dietary consumption of Ca. Recent experimental ev-
idence confirms this hypothesis. The expression of CLDN2
and of three TJ protein mRNAs (TJP 1,2,3 corresponding
to ZO 1,2,3) in duodenum were observed to increase with
age of the hen (12e23 wks) and to be highest in mature
hens (Gloux et al., 2019). CLDN2 expression also increases
in the jejunum between 17 and 23 wks of age and was
slightly higher during the final period of shell formation
(Gloux et al., 2020a). In contrast, CLDN10 decreased and
exhibited a higher level of expression in ileum at sexual
maturity (Gloux et al., 2019). In aged hens, the expression
of CLDN2 and of the anchoring protein (TJP3) were
downregulated, as was the VDR gene, suggesting a
decreased efficiency in paracellular Ca transfer with
increased age (Gloux et al., 2020b). Expression of other
candidates involved in the paracellular pathway, claudin 1
and 12, JAM and OCLN, were not affected by sexual
maturity nor by age of the hens (Gloux et al., 2020b).
In conclusion, numerous ionic transporters have been
now identified in the hen intestine, but no hierarchy has yet
been established on the relative roles of these candidates,
within a family or between families of a particular ionic
transporter (channel, pump, or exchanger). The role of
vitamin D metabolites on expression of TRPV, calbindin
and PMCA for the transcellular pathway and of some
CLDNs and TJPs for the paracellular transfer is demon-
strated in hens but remains to be characterized for addi-
tional proteins involved in calcium absorption.
32.6 Medullary bone
32.6.1 Structure and composition
One of the most relevant physiological adaptations that
female birds have developed to facilitate an adequate sup-
ply of calcium for eggshell mineralization is the develop-
ment of medullary bone, which can be more easily resorbed
to release calcium (Dacke et al., 1993, 2015; Nys and
Le Roy, 2018). Medullary bone (Figure 32.9) is a special
type of bone formed within the marrow cavities of long
bones of female birds during reproduction (Bloom et al.,
1941, 1958; Van de Velde, 1984b, 1985; Whitehead, 2004;
Kerschnitzki et al., 2014; Rodrõguez-Navarro et al., 2018).
The formation of medullary bone starts about 2 weeks
before laying of the first egg at sexual maturity and is
associated with higher levels of plasma estrogen and
vitamin D active metabolite (Figure 32.5). High-estrogen
levels produce a dramatic change in bone biology,
causing osteoblast function to switch from producing
cortical and trabecular bone to forming medullary bone
(Hudson et al., 1993; Whitehead, 2004). Medullary bone
partially fills the marrow space (endosteal cavities) of long
bones (tibia, humerus, femur), causing a 20% increase in
skeletal weight before the commencement of egg laying.
However, during the laying period, osteoclasts continue to
resorb cortical and trabecular bone, resulting in a progres-
sive reduction of the amount of structural bone and
increased bone porosity (Figure 32.10). The loss of struc-
tural bone is associated with a higher accumulation of
medullary bone, therefore the total amount of bone is
maintained at a nearly constant level. Nevertheless, the loss
of cortical bone induces a general weakening of the skel-
eton over the intensive laying period, causing hens to suffer
a high incidence of bone deformation and fractures (espe-
cially in the keel); this is a mayor welfare problem that also
has an important economic impact on egg producers
(Fleming et al., 2006). The medullary bone is deposited by
osteoblasts and represents about 11.7% of total bone cal-
cium. It is a nonstructural type of woven bone consisting of
a system of bone spicules that grow out from endosteal
surfaces and may completely fill the marrow spaces (Dacke
et al., 1993). Thus, medullary bone has no major mechan-
ical function and is distinct from the cancelous bone, which
is concentrated toward the metaphysis/epiphysis and has a
mechanical functionality dependent upon the integrity of
the intact long bone structure.
Laying
Non-Laying
FIGURE 32.9 Images showing the presence of medullary bone in hens
by comparing transverse section of the tibia of nonlaying and laying hens.
Image courtesy of the Roslin Institute R(D)SVS, University of Edin-
burgh, Scotland.
830 PART | V Endocrine theme
20. Cortical bone is constituted by aligned collagen fibrils
mineralized by apatite crystals oriented with their c-axis
parallel to the fibrils. In contrast, medullary bone is less
organized. It is formed by isolated mineral spicules or
trabecula. The spicules contain osteocytes and are sur-
rounded by a large number of osteoblasts and osteoclasts.
Internally, these spicules contain collagen fibers that run in
all directions. Apatite crystals of very small size cover the
collagen fibrils and mineralize an extracellular organic
matrix in the interfibrillar space consisting mainly of non-
collagenous proteins, glycoproteins, and proteoglycans
(Ascenzi et al., 1963; Bonucci and Gheraldi, 1975). This
mineralization consists of ribbon-shaped apatite crystals
distributed in separated bundles or foci that resemble the
early stages of embryonic bone mineralization
(Figure 32.10).
The mineral part of medullary bone is made of nano-
crystalline carbonate-apatite (calcium phosphate), similar to
cortical bone (Ascenzi et al., 1963). However, it also con-
tains a small fraction of calcite (CaCO3) as a separated
mineral phase (Lörcher and Newesely, 1969). The presence
of other more soluble and reactive mineral phases such as
calcite and possibly noncrystalline mineral phases (amor-
phous CaCO3 or calcium phosphate) could explain the
extremely high reactivity of this form of labile bone and its
main functionality as a source of calcium for the rapid
calcification of the eggshell. Its solubility is at least 30-fold
greater than that of cortical bone (Dominguez-Gasca et al.,
2019). The high solubility of medullary bone mineral is due
to its greater surface area, lower crystallinity, greater
carbonate content, and organic matrix composition. The
high-mineral solubility, together with its intense vascular-
ization and spicule concentration of bone cells, explains
why medullary bone can be metabolized at a much higher
rate than cortical bone. While cortical bone turnover can
take several months, medullary bone is turned over in only
three days (Van de Velde, 1984b, 1985). Moreover, the
composition of its organic matrix is believed to favor the
rapid mineralization/demineralization of medullary bone.
Medullary bone cell activities are synchronized with the
24-hour egg-laying cycle (Figure 32.11). During eggshell
calcification, there is an intense osteoclastic resorption of
medullary bone, followed by an intense osteoblastic ac-
tivity that forms new medullary bone before the beginning
of the next cycle of eggshell formation (Van de Velde,
1984b, 1985). In laying hens, the medullary bone is in
continuous activity throughout every stage of the egg-
laying period, although osteoclast activity increases dur-
ing calcification of the shell. Medullary bone serves as a
calcium reservoir for eggshell calcification when calcium
from the diet is exhausted (during the night) (Dacke et al.,
1993; Van de Velde, 1985; Whitehead, 2004). It can be
mobilized to provide 40% of the required calcium to the
eggshell daily, or up to 60% when hens are fed a low-
calcium diet. Taylor and Moore (1954) showed that hens
on a calcium deficient diet can mobilize up to 38% of
skeletal calcium before egg laying ceases. Regarding its
mechanical properties, even though it is less dense and
structurally weaker than cortical bone, medullary bone still
contributes significantly to the overall strength of bone
FIGURE 32.10 (A) Scanning electron microscope image of the tibia cross-section showing cortical bone (right) with large resorption centers and
medullary bone (left) formed by isolated mineral trabecula. (B) Transmission electron microscopy (TEM) image of cortical bone showing oriented apatite
crystals mineralizing a collagen matrix. (C) TEM image of medullary bone showing bundles of randomly oriented apatite crystals mineralizing a non-
collagen matrix. Scale bars: (A) 100 mm; (BeC) 200 nm (Dominguez-Gasca et al., 2019).
Mechanisms and hormonal regulation of shell formation Chapter | 32 831
21. (Fleming et al. al., 2006; Rodrõguez-Navarro et al., 2018).
In fact, the amount of medullary bone mineral has a large
genetic correlation with tibia bone breaking strength (Dunn
et al., in press, 2020). Additionally, the large amount of
medullary bone that accumulates near the endosteal bone
surfaces can also protect cortical bone against osteoclast
resorption.
32.6.2 Regulation of medullary bone formation
and resorption
32.6.2.1 Induction and maintenance of
medullary bone by sex steroid hormones
Avian medullary bone probably represents the most
estrogen-sensitive of all known vertebrate bone types, as
gonadal steroids are absolutely essential for the induction
and maintenance of medullary bone in egg-laying birds
(Simkiss, 1967; Whitehead, 2004; 2015; Squire et al.,
2017). In laying hens, the secretion of androgens and es-
trogens increases at sexual maturity. These gonadal hor-
mones have a synergistic action on medullary bone
formation, which can also be induced in male birds by
estrogen treatment (Whitehead, 2004; Simkiss, 1967;
Squire et al., 2017). Estrogens induce differentiation of
endosteal cells to form osteoblasts and decrease the number
of osteoclasts and their activity on the endosteal surface
(Ascenzi et al., 1963; Miller and Bowman, 1981). Estrogen
receptors are present in medullary bone osteoblasts (Ohashi
et al., 1991) and osteoclasts (Oursler et al., 1993). In laying
hens fed a calcium deficient diet, the medullary bone shows
a large increase in the osteoblast population, the osteoclasts
being substituted by osteoblasts on the trabecular surface
(Zambonin-Zallone and Mueller, 1969). In addition, the
percentage of medullary bone in the skeleton increases
because of the depletion of cortical bone, which reveals the
priority for medullary bone in reproductive hens (Dacke
et al., 2015). However, the balance between the two types
of bone and severity of bone loss depends on the duration
of a calcium and vitamin D deficiency; clearly a reduction
in dietary calcium and vitamin D has negative effects on
bone mineralization (Dacke et al., 2015).
Bone is a metabolically active calcified tissue in a
constant state of remodeling. Bone mineralization/demin-
eralization is regulated by a complicated array of feedback
processes under the control of specialized hormones related
to calcium homeostasis (PTH, vitamin D). It has been well
established that vitamin D contributes to bone mineraliza-
tion in mammals (Pike et al., 2014; Christakos, 2014,
2019), partially by the activation of osteoblastic activity
and also by the induction of bone protein synthesis.
Vitamin D is clearly a key element for bone mineralization
in growing chickens, but also in hens, even if medullary
bone formation appears to be a priority relative to cortical
bone in sexually mature hens. Vitamin D will globally
contribute to the supply of calcium and phosphorus to
facilitate bone mineralization, and more specifically, it will
stimulate the synthesis of certain bone matrix proteins, as
demonstrated for osteocalcin and osteopontin (OPN).
Osteocalcin (OC) (bone g-carboxy-glutamic acid protein) is
the most abundant noncollagenous protein associated with
the mineralized matrix of bone (Price, 1985). It has the
ability to bind calcium, and it shows adsorption affinity for
hydroxyapatite (Hauschka et al., 1989). This non-
collagenous protein also plays a role in bone resorption
because of its implication in the differentiation of osteo-
clasts (Ishida and Amano, 2004). OPN (SPP1) is a glyco-
sylated, highly phosphorylated protein initially identified in
bone and also present in eggshell (Chien et al., 2008;
Hincke et al., 2008). In bone, SPP1 is considered to in-
fluence the migration and maturation of osteoclast pre-
cursors, the attachment of osteoclasts to the mineral phase
of the bone, and osteoclast activity. 1,25(OH)2D3 stimu-
lates OC (Lian and Stein, 1992) and OPN (Noda et al.,
1990) synthesis in osteoblast cell culture by binding to
specific vitamin D-response promoter elements to enhance
their gene transcription. However, OC synthesis, in contrast
to calbindin, is modulated by, rather than dependent upon,
vitamin D since there is substantial OC synthesis in vitamin
D-deficient chicks. It has been shown that medullary bone
hrs a er
ovula on
Plasma
Pi
(mg/l)
50
40
30
20
Urinary
phosphate
(μmol/min)
10
5
0
4 8 10 12 22
16
0
Eggshell forma on
Mineral resorp on
Matrix forma on
Mineral
accre on
FIGURE 32.11 Plasma levels of inorganic phosphorus and urinary
excretion of phosphorus during the laying cycle or in cokerel (dashes).
Shell formation occurred 10e22 h after yolk ovulation in the oviduct.
Laying hens were fed 3.5% dietary calcium, as fine (continuous line)or
coarse particles of calcium carbonate (dotted line). Urinary calcium was
sampled in hens fed fine particle calcium (3.5%). During the night (no food
intake), the hen mobilize more Ca from the bone when fed fine particle
compared to coarse one (Nys and Le Roy, 2018).
832 PART | V Endocrine theme
22. contains multiple isoforms of bone sialoprotein (BSP),
OPN, osteonectin, OC, and dentin matrix protein-1 (Wang
et al., 2005). Another particularity of medullary bone
compared to cortical bone is the presence of large amounts
of a keratan sulfate (KS) proteoglycan (KSPG). The core
protein of KSPG is BSP (Hadley et al., 2016), which plays
a key role in bone mineralization and remodeling (Staines
et al., 2012). It was shown previously that KS is the major
proteoglycan in medullary bone rather than chondroitin
sulfate (Fisher and Schraer, 1980; Hunter and Schraer,
1983). Therefore, Hadley et al. (2016) have proposed that
plasma levels of KS are a specific biomarker for medullary
bone metabolism and have demonstrated that it indeed
fluctuates in synchrony with the egg-laying cycle.
In hens, matrix formation within medullary bone can be
induced by sex steroids regardless of the vitamin D status
of chicks, but its mineralization is observed only when
vitamin D3 is administered together with the gonadal ste-
roids (Takahashi et al., 1983). It is likely that vitamin D is a
key element which favors osteoblast activity in both
cortical and medullary bone; however, the balance between
both tissues is modulated by sex steroids, possibly indi-
rectly, through their effects on the general parameters of
bone physiology(vasculature and larger exchange surface
area) (van de Velde et al., 1984b; Dacke et al., 2015). In
addition, it has been demonstrated that estrogens stimulate
OC secretion in response to 1,25(OH)2D3 in osteoblast-like
cells by increasing VDR expression, which supports the
involvement of vitamin D in medullary bone formation. OC
is predominantly synthesized by osteoblasts, is partially
released into the circulation and has a rapid turnover
(Figures 32.5 and 32.6) (Nys et al., 1986; Nys, 1993). It
therefore reflects osteoblast synthesis and provides an index
of bone turnover in humans (Delmas, 1992). Its plasma
variation has been explored in hens in various physiological
situations associated with large changes in plasma
1,25(OH)2D3 (Figures 32.5 and 32.6). Unexpectedly, in
immature pullets treated with gonadal steroids or in laying
hens, plasma OC is lower than that of immature pullets, in
contrast to the higher levels of plasma 1,25(OH)2D3
(Figure 32.5). In hens, daily changes in plasma OC levels,
however, parallel those in plasma 1,25(OH)2D3 when
inducing variation in bone activity by feeding hens with
low-calcium diet or by suppressing shell formation in hens
fed normal high-calcium diet (Figure 32.6). It increases
during the period of shell formation in hens fed a low-
calcium diet with a very high level of 1,25(OH)2D3 and
decreases in hens laying shell-less eggs possessing lower
plasma 1,25(OH)2D3 levels Nys (1993). It is further stim-
ulated in hens fed a low-calcium diet when bone is highly
implicated in shell formation. Plasma OC corresponds to
the period of bone matrix synthesis, but also coincides with
the period of most active bone resorption (van de Velde
et al., 1984b). Therefore, hen plasma OC levels reflect
osteoblastic activity only during the daily laying cycle but
not during the long-term changes that occur with sexual
maturity. These observations might be because of large
mobilization of OC in the tissue during intensive bone
formation, it being trapped in bone and no longer secreted
in the plasma. It is of interest to note that estrogens stim-
ulate OC secretion in response to 1,25(OH)2D3 in
osteoblast-like cells by increasing VDR expression.
32.6.2.2 Role of parathyroid hormone in daily
mobilization of medullary bone
In hens, medullary bone is considered to be a labile calcium
reservoir because of the daily osteoblast/osteoclast remod-
eling which supplies calcium for shell formation during the
period when no dietary calcium is available (Hurwitz,
1965; Dacke et al., 2015; Whitehead, 2004). Eggshell
formation takes place mainly during the night, when the
hens have to mobilize calcium from bone since dietary
calcium has been exhausted. Thus, medullary bone serves
as a calcium reservoir for eggshell calcification and buffers
the supply of calcium in response to its cyclical require-
ment. Use of large CaCO3 particles, which slows down
intestinal calcium solubilization and provides a higher
calcium intestinal supply throughout the night, reduces
bone mobilization (Figure 32.11). This nutritional approach
is intensively used in practical conditions for improving
eggshell quality (strength and thickness). The large
resorption of medullary bone during the period of shell
formation is clearly demonstrated by the huge increase in
plasma phosphorus and its excretion in urine observed in
hens from 10 to 22 h postovulation (Figure 32.11; Prashad
and Edwards, 1973), which coincides with the period of
shell deposition Wideman et al. (1987). These changes are
abolished when hens are laying shell-less eggs. van de
Velde et al. (1984a) reported that bone resorption was
increased ninefold during shell formation due to an increase
in active osteoclasts and in the resorbing surface per active
osteoclast (Figure 32.11). At the same time, osteoblasts
deposited some matrix protein, contributing to bone ac-
cretion that was also activated twofold to renew the med-
ullary bone. There is a lot of evidence demonstrating that
this diurnal regulation of bone resorption is under the
control of PTH (van de Velde et al., 1984a; Kerschnitzki
et al., 2014; Dacke et al., 2015). PTH secretion is increased
during the period of shell formation (van de Velde et al.,
1984a; Singh et al., 1986; Kerschnitzki et al., 2014)
because of lower ionized calcium in the plasma. PTH
stimulates bone resorption acutely, as described in the
previous section describing the role of PTH in birds. It is
noteworthy that FGF23 (phosphatonin) is expressed in
medullary bone, in synchrony with the egg-laying cycle
(Hadley et al., 2016; Gloux et al., 2019). This phosphate-
mic hormone is produced by osteocytes and plays a key
Mechanisms and hormonal regulation of shell formation Chapter | 32 833
23. role in maintaining phosphate homeostasis, as described in
the section of this review that discusses the role of hor-
mones in Ca metabolism in hens.
32.7 Uterine secretions of Calcium
32.7.1 Mechanisms of ionic transfers
Eggshell formation takes place in the uterine segment of
the oviduct (shell gland). Daily shell formation is associ-
ated with a massive transfer of calcium and bicarbonate
into the lumen of the uterus during a short period (12hrs).
In chickens, the mean rate of accumulation of CaCO3
is 0.33 g/h between the period 10 to 22 h following
ovulation and entry of the yolk into the oviduct (Eastin and
Spaziani, 1978a; Nys and Guyot, 2011) this results in
deposition of more than 6g of shell mineral. Calcium
transfer follows a favorable positive (blood/uterine fluid)
electrical potential difference (Pearson and Goldner, 1973,
1974; Bar, 2008), but is against a large and unfavorable
Ca2þ
gradient, since the level of ionized calcium in plasma
(1.2 mM) is lower than its concentration in the uterine fluid
(6 to 10 mMddepending on the stage of eggshell calcifi-
cation) (Arad et al., 1989; Nys et al., 1991). The trans-
cellular Ca2þ
transport system has some similarities with
mechanisms of Ca absorption in the intestine, even if the
fluxes are inversed (Table 32.1). Calcium entry occurs in
three steps (Figure 32.12), as reviewed by numerous au-
thors (Hurwitz, 1989a; Nys et al., 1999; Bar, 2009): (1)
passive entry of Ca2þ
from plasma into the uterine cells via
Ca channels; (2) its transfer through the cytosol bound to
the Ca-binding protein, calbindin 28k; and (3) its extrusion
by a calcium pump and Na/Ca exchanger. Many trans-
cellular transporters of additional ionic species (HCO3,
Naþ
, Kþ
, Cl
, Hþ
) participate in the process of calcium
secretion and in the maintenance of cellular ionic homeo-
stasis (Eastin, W.C. and Spaziani, E., 1978a, 1978b; Jon-
chere et al., 2012; Brionne et al., 2014; Sah et al., 2018;
Zhang et al., 2020), as observed at the intestinal level (Bar,
2009; Gloux et al., 2019). However, the additional contri-
bution of a paracellular pathway for Ca is probably minor,
even if this issue remains controversial for the uterus (Bar,
2009; Nys and Le Roy, 2018). Ca2þ
secretion in the uterine
lumen of hens shows particular features. Firstly, it has been
plasma
uterine
lumen
Ca2+
Na+
Ca2+
2H+
Ca2+
2H+
Ca2+
Ca2+
EnRe
N
ATP2B1/2 ATP2A2/3
SLC8A1/3
TRPV6, 2, 4 6 TRPC1
ITPR1/2/3
6,5pH7
CALB1
Calcium
?
PKP2, TRPM7, CACNA1D 1E 1H
Paracellular
pathway
1,25(OH)2D3 VDR Vitamin D Receptor
apical
basal
Tight juncon
Ca2+
Vesicles
ER
ER
Estrogen Receptor
Estrogen
Calcium parcles
EOM +
transporters
Na+
SCNN1A
SCNN1B
SCNN1G
KCNJ2
KCNJ15
KCNJ16
KCNH1
K+
Ca2+ + HCO3
-
CaCO3
H+
FIGURE 32.12 General model describing uterine ion transporters through the uterine cells of the laying hen. All symbols are defined in the legend part
(right). EnRe, endoplasmic reticulum; EOM, extracellular organic matrix; N, nucleus. Data are compiled from Jonchere et al. (2012); Brionne et al.
(2014); Nys and Le Roy (2018).
834 PART | V Endocrine theme
