Received: 31 October 2018 | Revised: 16 April 2019
DOI: 10.1002/jez.b.22892
RESEARCH ARTICLE
Alpha‐keratin and corneous beta protein in the
parakeratinized epithelium of the tongue in the
domestic goose (Anser anser f. domestica)
Kinga Skieresz‐Szewczyk1
Hanna Jackowiak1
1
Department of Histology and Embryology,
Faculty of Veterinary Medicine and Animal
Science, Poznan University of Life Sciences,
Wojska Polskiego, Poznan, Poland
2 is characterized by the presence of cell nuclei in the cells of the cornified layer. This
Institute of Materials Research and Quantum
Engineering, Faculty of Technical Physics,
Poznan University of Technology, Piotrowo,
Poznan, Poland
Correspondence
Kinga Skieresz‐Szewczyk, Department of
Histology and Embryology, Faculty of
Veterinary Medicine and Animal Science,
Poznan University of Life Sciences, Wojska
Polskiego 71C, 60‐625 Poznan, Poland.
Email: kinga.skieresz-szewczyk@up.poznan.pl
The peer review history for this article is
available at https://publons.com/publon/10.
1002/jez.b.22892/
1 | INTRODUCTION
Mammals, birds, and reptiles adapt to the diverse environment due to
the formation of different cornified epidermal structures (scales, claws,
feather, beak, carapace; Alibardi, 2003, 2006, 2007, 2009a, 2009b;
158 | © 2019 Wiley Periodicals, Inc.
| Accepted: 22 May 2019
| Tomasz Buchwald2 | Mirosław Szybowicz2 |
Abstract
The parakeratinized epithelium is a common epithelium in the oral cavity in birds and
epithelium covers almost the entire dorsal surface of the tongue in the domestic
goose apart of the lingual nail and conical papillae. So far no study has identified the
molecular proteins alpha‐keratin (IF‐keratin) and/or corneous beta protein (CBP),
which are responsible for keratinization or cornification processes in the
parakeratinized epithelium of domestic geese. The study was performed using
immunohistochemical (IHC) methods to identify alpha‐keratin. The innovative
method of Raman microspectroscopy was used to determine the presence of CBP
and specify their percentage in epithelial layers of the parakeratinized epithelium.
The results revealed that alpha‐keratin is present in the whole parakeratinized
epithelium. A strong staining reaction was detected in the basal and intermediate
layers and a less strong staining reaction in the cornified layer. Raman microspectro-
scopy analysis confirmed the presence of alpha‐keratin and demonstrated that its
percentage decreases from the basal layer to the cornified layer. The Raman
microspectroscopy technique revealed the occurrence of CBP in the parakeratinized
epithelium and demonstrated that the percentage of this protein increases from the
basal layer to the cornified layer. Performed analysis determines that parakeratinized
epithelium undergoes cornification. However, the lower percentage of CBP in the
cornified layer of parakeratinized epithelium than in orthokeratinized epithelium
points to the fact that parakeratinized epithelium has a weaker protective function.
KEYWORDS
alpha‐keratin, birds, corneous beta protein, parakeratinized epithelium, Raman
microspectroscopy, tongue
Dhouailly, 2009; Sawyer & Knapp, 2003). The cornification process in
birds and reptiles involves gradual accumulation in the keratinocytes of
the specific corneous beta protein (CBP), which accumulate around the
alpha‐keratin (IF‐keratin) and creates hard corneous material in the
cornified layer (Alibardi, 2002, 2004, 2007, 2009a; Sawyer & Knapp,
wileyonlinelibrary.com/journal/jezb J Exp Zool (Mol Dev Evol). 2019;332:158–166.
SKIERESZ‐SZEWCZYK ET AL.
2003; Walker & Rogers, 1976). In mammals, the epidermis and cornified
epidermal structures undergo cornification process that involves the
accumulation in keratinocytes of different keratin (acidic and basic
keratin) and keratin‐associated proteins (KAPs), mainly fillagrin and
loricrin (Bragulla & Homberger, 2009; Moll, Divo, & Langbein, 2008;
Steven & Steinert, 1994). Genes encoding KAPs in human are located in
chromosome 1, locus called epidermal differentiation complex (EDC;
Mischke, Korge, Marenholz, Volz, & Ziegler, 1996). Studies by Strasser
et al. (2014) reveals that in reptiles and birds, genes encoding the CBP,
as in mammals, are placed in an EDC locus. CBP genes are located in a
single locus in lizards and in one main locus and in a few additional loci
in birds and reptiles (Dalla Valle et al., 2010; Gregg & Rogers, 1986).
Translocation of some CBP genes from the EDC locus in birds and
turtles revealed its relationship to the formation of appendages, such as
feather and carapaces (Holthaus et al., 2016, Holtahus, Eckhart, Dalla
Valle & Alinardi, 2019). However, loci with genes encoding alpha‐keratin
are distant and unrelated phylogenetically with CBP genes (Holthaus
et al., 2019).
The keratinized epithelium not only covers the skin and form
skin appendages but also covers mucosa of the oral cavity in
vertebrates (Herrel, Canbek, Ozelmas, Uyanoglu, & Karakaya, 2005;
Homberger & Brush, 1986; Iwasaki & Miyata, 1989, 1990; Iwasaki,
Asami, & Chiba, 1997; Jackowiak & Godynicki, 2005; Jackowiak,
Skieresz‐Szewczyk, Godynicki, Iwasaki, & Meyer, 2011; Jackowiak,
Skieresz‐Szewczyk, Kwieciński, Trzcielińska‐Lorych, & Godynicki,
2010; Skieresz‐Szewczyk & Jackowiak, 2016). The lingual mucosa in
birds as in mammals is covered with two different types of the
keratinized epithelia, that is ortho‐ and parakeratinized epithelium
(Iwasaki et al., 1997; Jackowiak & Godynicki, 2005; Jackowiak et al.,
2010, 2011; Pedersen & Reibel, 2014; Sawaf, Ouhayoun,
Shabana, & Forest, 1990; Skieresz‐Szewczyk & Jackowiak, 2016;
Skieresz‐Szewczyk, Jackowiak, & Ratajczak, 2014). The presence of
those two types of epithelia correlates with the type of collected food,
the way of food intake and type of food transport to the esophagus
(Jackowiak et al., 2011; Skieresz‐Szewczyk & Jackowiak, 2016;
Skieresz‐Szewczyk et al., 2014). Generally, the orthokeratinized
epithelium is present in places that are involved in food collection
such as mechanical papillae of the tongue in mammals and birds and is
ventrally positioned in the cornified layer of the lingual nail on lingual
apex in birds (Iwasaki & Miyata, 1989, 1990; Iwasaki et al., 1997;
Jackowiak & Godynicki, 2005; Jackowiak et al., 2010, 2011; Skieresz‐
Szewczyk & Jackowiak, 2016). The parakeratinized epithelium is
located on the top of the fungiform papillae and gingiva in mammals
and on the dorsal surface of the tongue in birds, where is functionally
linked to food transport (Jackowiak et al., 2011; Sawaf et al., 1990). In
the domestic goose, due to over‐tongue transport, the parakeratinized
epithelium covers the whole dorsal surface of the tongue, from the
apex to the root of the tongue (Jackowiak et al., 2011). Orthoker-
atinized epithelium in the domestic goose, as in other birds, covers the
mechanical papillae of the tongue and forms the cornified plate of the
lingual nail (Jackowiak et al., 2011).
The epithelium of the lingual mucosa in birds, unlike in mammals, is
built from three layers, that is, the basal, intermediate and keratinized
| 159
layer (Iwasaki et al., 1997; Jackowiak et al., 2011; Skieresz‐Szewczyk &
Jackowiak, 2016; Skieresz‐Szewczyk et al., 2014). The characteristic
features of the bird's ortho‐ and parakeratinized epithelium is the
presence of two zones in the intermediate layer and lack of
keratohyalin granules in the epithelial cell cytoplasm, which is typical
for keratinized epithelium in the mammals' oral cavity (Adams, 1976;
Iwasaki & Miyata, 1989, 1990). The parakeratinized epithelium in
birds, as in mammals, is characterized by the presence of a cell nucleus
with condensed chromatin in the cell cytoplasm of the keratinized
layer (Pedersen & Reibel, 2014; Sawaf et al., 1990).
The previous immunohistochemical (IHC) studies of the ortho-
keratinized epithelium of the tongue in birds reveal the presence of
alpha‐keratin and CBP (former name beta‐keratin; Carver & Sawyer,
1989; Carver, Knapp, & Sawyer, 1990; Homberger & Brush, 1986).
Our own research has shown that Raman microspectroscopy offers
an alternative method to detect CBP, in the absence of avian‐specific
antibodies (Skieresz‐Szewczyk, Jackowiak, Buchwald, & Szybowicz,
2017). Raman spectroscopy is an effective method for analysis of the
secondary structure of proteins such as alpha‐helix, beta‐sheet, or
beta‐turn, at the microstructural level (Buchwald et al., 2012; Pelton
& McLean, 2000; Rizzo et al., 2006; Lefevre, Rousseau, & Pezolet,
2007; Church, Poole, & Woodhead, 2010; Jastrzębska et al., 2016).
Moreover, this method allows one to indicate the spatial distribution
of proteins in a biological probe visualized on Raman spectral maps.
The present study aims to determine for the first time, whether
the parakeratinized epithelium of the tongue in domestic goose, like
the previously described orthokeratinized epithelium, undergoes
cornification processes. To answer this question were performed IHC
analysis to identify alpha‐keratin and Raman microspectroscopy
method to detect CBP. Therefore, in the present study Raman
spectroscopy was also used to identify the secondary structure of the
proteins as well as determining the percentage share of a
alpha‐keratin and CBP in the individual layers of the epithelium.
The obtained results will be used for further comparative studies on
various keratinized epithelia of the tongue in birds as well as
cornified structures of the epidermis in birds and reptiles.
2 | M A T E R I A L S AN D M E T H O D S
The study proceeded on the eight tongues of adult domestic goose
(9‐month old, the average weight of 5.5 kg) collected from a local
slaughterhouse. According to Polish law and the EU directive no
2010/63/EU the present study does not require the approval of the
Local Ethical Committee for Experiments on Animals in Poznan.
2.1 | Microscopic and IHC methods
The three dissected tongues were rinsed in saline and immersed for
24‐hr in 4% neutralized formalin. After a fixation period, the
tissue samples from the dorsal surface of the body of the
tongue were collected, dehydrated in a series of increasing
concentration of ethanol (70–96%), and routinely embedded in
160 | SKIERESZ‐SZEWCZYK
Paraplast® (Sigma‐Aldrich, Germany). Paraplast blocks were cut into
serial sections of 4.5–5 μm. Tissue sections were stained using the
Masson‐Goldner trichromie histological staining technique (Romeis,
1989). Tissue samples for immunohistochemistry staining with
cytokeratin clone AE1/AE3 (Dako; dilution 1:50) proceeded accord-
ing to the protocol by Skieresz‐Szewczyk et al. (2017).
2.2 | Raman microspectroscopic analysis
The five tongues of domestic geese were immersed in 4% neutralized
formalin. Before Raman spectroscopy measurements the specimens were
rinsed in distilled water. The samples were analyzed at room
temperature. The Raman spectroscopy measurements were carried out
by inVia Renishaw microscope equipped with a laser emitting 785 nm
near‐infrared wavelength. During measurements, the laser beam was
automatically focused on the sample through a ×20 microscope objective
(Leica) with a numerical aperture of 0.4. The air‐cooled Rencam CCD
Camera detector and 1,200 l/mm diffraction grating were used. At the
beginning of each measurement session, the spectral data were calibrated
with the use of Raman band at 520.7 cm −1
of a silicon internal reference
sample. Raman spectra were collected in the spectral range from 200 to
−1
1,800 cm . The background in Raman spectra of alpha‐keratin
(IF‐keratin) and CBP, presented in Figure 3, was subtracted by using
the polynomial curve. However, the spectra of Raman maps were
analyzed without the background removed. The integrated intensity of
Raman bands in amide I region (from 1,590 to 1,710 cm−1) were obtained
by the curve‐fitting process in single spectra by use of WiRE 3.4
(Renishaw) software. In this process, the combination of Gaussian and
Lorentzian functions was used to obtain the integrated intensity of
Raman bands. The integrated intensity were used to detect the relative
quantity of particular secondary structure (random coil, alpha helix, beta
sheet, and beta turn) of proteins in tongues. Quantitative changes in
proteins secondary structure were presented using the Raman spectral
maps. Before Raman maps measurements the tongues were intersected.
Raman maps were collected on the cross section of tongues (on the cut
surface). The Raman spectral maps were collected in rectangular area of
250 µm × 1,200 µm with a single analysis of each data point (steps of
50 µm in the x and y directions). The epithelium on the dorsal surface of
the lingual body was analyzed in detail, with 150 Raman spectra collected
during measurement of a single map. In this study, the Raman maps with
a percentage distribution of proteins for the cornified, intermediate and
ET AL.
basal layers in the parakeratinized epithelium were presented for one
exemplary representative tongue only.
3 | RESULTS
3.1 | Microscopic and IHC analysis
The parakeratinized epithelium of the dorsal surface of the lingual
body in the domestic goose consisted of three layers: basal,
intermediate, and cornified layer (Figures 1,2). The basal layer was
built of elongated epithelial cells with oval cell nucleus with one to
two nucleoli (Figure 2a). The epithelial cells in the intermediate layer
were arranged into two zones. In the lower zone of the intermediate
layer, all cells were polygonal in shape and had horizontally located
oval nucleus with one to two nucleoli (Figure 2a). Cells in the upper
zone of the intermediate layer were polygonal and slightly flattened
with flat cell nucleus with one nucleoli (Figure 2a). Cell cytoplasm in
both, lower and upper zone of the intermediate layer, after Masson‐
Goldner trichrome staining, had a uniformly pink color (Figure 2a).
Epithelial cells in the cornified layer were flat and had cell nucleus
with condensed chromatin (Figure 3c). The Masson‐Goldner staining
showed the diverse coloration of the cell cytoplasm in the cornified
layer. The most outer cornified cells had pale pink color of cell
cytoplasm and cells located underneath had an intensive red‐colored
cell cytoplasm (Figure 3c). The cells on the surface exfoliate one by
one (Figure 2a).
The cell cytoplasm of all epithelial layers revealed the IHC
positive staining reaction with alpha‐keratin (Figure 2b). The strong
staining reaction was visible in lower layers of the epithelium, namely
in the basal and intermediate layer (Figure 3a,b). The less strong
staining reaction was observed in the cornified layer (Figure 3d). In
addition, different staining reaction was detected in the epithelial
cells of the cornified layer. The most outer epithelial cells had strong
staining reaction while the epithelial cells located below had weak
staining reaction (Figure 3d).
3.2 | Raman microspectroscopy analysis
Raman spectroscopy was applied to identify the secondary structure
of proteins in the parakeratinized epithelium of the lingual body of
the tongue of domestic goose. Figure 4a shows an example of a
F I G U R E 1 Dorsal view on the tongue in
the domestic goose. Dotted line mark area
of the apex (a), body (b), lingual
prominence (Lp) and root of the tongue (R)
covered with parakeratinized epithelium.
Scale bar = 1 cm [Color figure can be
viewed at wileyonlinelibrary.com]
SKIERESZ‐SZEWCZYK ET AL. | 161

F I G U R E 2 (a) Cross‐section through

the parakeratinized epithelium of the
dorsal surface of lingual body. Scale
bar = 20 µm. (b) Immunohistochemical
staining of the alpha‐keratin in the
parakeratinized epithelium of the dorsal
surface of the lingual body. Scale
bar = 20 µm. Bl, basal layer; Cl, cornified
layer; LInt, lower zone of the intermediate
layer; Lp, lamina propria; UInt, upper zone
of the intermediate layer [Color figure can
be viewed at wileyonlinelibrary.com]

F I G U R E 3 (a) Higher magnification of the immunohistochemical staining of the basal layer of the parakeratinized epithelium of the dorsal
surface of the lingual body. Scale bar = 50 µm. (b) Higher magnification of the immunohistochemical staining of the intermediate layer of the
parakeratinized epithelium of the dorsal surface of the lingual body. Scale bar = 50 µm. (c) Higher magnification of the cornified layer of the
parakeratinized epithelium of the dorsal surface of the lingual body. Black arrow points most outer pale pink epithelial cell. White arrow
indicates red colored epithelial cell. Scale bar = 50 µm. (d) Higher magnification of the immunohistochemical staining of the cornified layer of the
parakeratinized epithelium of the dorsal surface of the lingual body. Black arrow points most outer epithelial cell with strong staining reaction.
White arrow indicates epithelial cell with weak staining reaction. Scale bar = 50 µm [Color figure can be viewed at wileyonlinelibrary.com]

Raman spectrum collected in studied tissues. To obtain a percentage overlapping bands. Examples of the deconvolution of a spectrum
content of the type of secondary structure of proteins the collected in the cornified layer (b) and intermediate and basal layers
curve‐fitting process of Raman band related to amide I (labeled in (c) is presented in Figure 4. The bands corresponding to the alpha‐
spectrum in Figure 4a) was used to obtain the integrated intensity of helix and beta‐sheet conformations occur at 1,652 and 1,669 cm−1,
162 | SKIERESZ‐SZEWCZYK ET AL.

F I G U R E 4 An exemplary Raman spectrum of the proteins in (a) the parakeratinized epithelium of the dorsal surface of the lingual body
with marked of analyzed amide I band. Deconvolution of amide I band in Raman spectra collected in (b) cornified layer and (c) basal and
intermediate layer [Color figure can be viewed at wileyonlinelibrary.com]

respectively. In turn, the bands at 1,685 and 1,698 cm−1 are
associated with beta‐turn conformation. The band associated with
unordered secondary structure of proteins (random coil) is detected
at 1,638 cm−1. Raman spectrum of proteins provides additional
information about aromatic residues in the spectral region below
−1 −1
1,620 cm . The Raman bands near 1,604 and 1,615 cm correspond
to phenylalanine (Phe) and tyrosine (Tyr) ring modes, respectively.
The integrated intensity of bands obtained after analysis of amide
I band allows on accurate evaluates for alpha helix, beta sheet, beta
turn, and random coil percentage content in the analyzed paraker-
atinized epithelium. To determine the spatial changes in the relative
quantity of proteins in a particular type of conformation in cornified
layer, the intermediate and basal layer, Raman maps were used. The
local variations in proteins conformation give information about the
alpha‐keratin and CBP occurring in particular layers. Figure 5 shows
a cross‐section through the parakeratinized epithelium of the dorsal
surface of the body of the tongue (on the left side) and the maps
showing the percentage change of proteins content in a random coil,
beta sheet, beta turn and alpha helix conformation in the specified
layer of the epithelium (on the right side). The Raman spectral maps
were collected from the outer layer (cornified layer) to the lower
layers (the intermediate and basal layers) of the parakeratinized
epithelium. The distribution of alpha‐keratin in studied material is
showed by the distribution of proteins in an alpha helix conformation.
In turn, the distribution of CBP in tongue is presented by the sum of
proteins in beta sheet and also beta‐turn conformation. The colors in
the maps correspond to particular percentage content of secondary
structure of proteins. The color bar presents the assigning the color
to percentage content for each image. Based on the maps can be
concluded that beta‐sheet content change from 20% to 60% in the
SKIERESZ‐SZEWCZYK ET AL. | 163

F I G U R E 5 Immunohistochemical staining of alpha‐keratin in the parakeratinized epithelium of the dorsal surface of the lingual body (on the
left side) and maps with percentage distribution of proteins in cornified, intermediate and basal layers in the parakeratinized epithelium in
random coil, beta sheet, beta‐turn and alpha helix conformation and distribution of corneous beta protein and alpha‐keratin calculated by curve‐
fitting process in amide I band region of Raman spectra (on the right side). The color bar displays the percentage of proteins in a different
conformation. Red and blue colors correspond to the highest and the lowest percentage of proteins in each conformation, respectively
[Color figure can be viewed at wileyonlinelibrary.com]

cornified layer, and from 10% to 30% in basal and intermediate
layers, whereas the beta‐turn content change from 10% to 40% in
cornified layer, and from 10% to 30% in basal and intermediate
layers. The alpha helix content changes from 0% to 30% in the
cornified layer and from 20% to 60% in the basal and intermediate
layers. The random coil content change from 10% to 30% in the
cornified layer as well as in the basal and intermediate layers. The
sum of the beta sheet and beta‐turn conformations content changes
from 50% to 70% in the cornified layer and from 30% to 60% in the
basal and intermediate layers.
4 | D IS C U S S IO N
The cornification process of the stratified epithelia in birds
depends on the accumulation of alpha‐keratin and CBP (Alibardi,
2002, 2004, 2007; Carver & Sawyer, 1989; Carver et al., 1990,
2009; Moll et al., 2008; Sawyer & Knapp, 2003; Walker & Rogers,
1976). Alpha‐keratin is a 40–70 kDa protein with an alpha‐helical
secondary structure that forms intermediate filaments with a
diameter of 7–10 nm constituting part of the cytoskeleton of the
epithelial cells (Alibardi, 2004; Alibardi & Toni, 2004; Bragulla &
Homberger, 2009; Carver & Sawyer, 1989). CBP is a 8–30 kDa
protein in epithelial cells, characterized with beta‐sheet secondary
structure and forms filaments with a diameter of about 4 nm which
due to their structure are not classified as intermediate filaments
(Alibardi, 2004; Alibardi & Toni, 2004; Carver & Sawyer, 1989;
Fraser & Parry, 1996). Alpha‐keratin is present in
interfollicular epidermis, its forms the inner surface of claws, and
is also present in the orthokeratinized epithelium on the ventral
surface of the lingual apex in birds (Alibardi, 2007, 2009a, 2009b;
Skieresz‐Szewczyk et al., 2017). CBP fills the keratinocytes in the
thick corneous layer on the dorsal surface of the claws in birds and
the cornified plate of the lingual nail (Alibardi, 2009a, 2009b;
Skieresz‐Szewczyk et al., 2017).
The present combined IHC study and Raman spectroscopy
analysis, for the first time, identify alpha‐keratin and CBP in all
epithelial layers of the parakeratinized epithelium of the tongue in
birds. There were observed differences in the intensity of staining
with alpha‐keratin, particularly between epithelial layers. A strong
staining reaction was observed in the basal and intermediate layer
and medium staining reaction in the cornified layer.
Carver and Sawyer (1989) analyzed the tongue of the domestic
chicken. They used IHC to identify the presence of alpha‐keratin and
CBP in the epithelium of the dorsal surface of the tongue. However,
the authors do not define this epithelium as a parakeratinized
epithelium but as a stratified squamous epithelium. That study
reveals that the alpha‐keratin is present in the basal and superficial
layers. There is no alpha‐keratin in the intermediate layer whereas
CBP is present occasionally in the superficial cells.
The Raman microspectroscopy method used in the present study,
allows to determinine percentage distribution of the alpha‐keratin
and CBP in the parakeratinized epithelium. In the basal and
intermediate layer, the percentage of alpha‐keratin is about
38 ± 8% (between 20% and 60%), while in the cornified layer it is
the only about 20 ± 7% (between 0% and 30%). These results explain the
164 | SKIERESZ‐SZEWCZYK ET AL.

weaker IHC staining reaction in the cornified layer than in the T A B L E 1 Spatial distribution of the alpha‐keratin (IF‐keratin) and
intermediate and basal layers of the parakeratinized epithelium. corneous beta protein and their percentage amount in particular
Deconvolution of the band assigned to amide I allowed an layers of the para‐ and orthokeratinized epithelium of the tongue in
the domestic goose obtained by IHC staining and Raman micro-
evaluation of the relative quantity of the CBP in the beta sheet and
spectroscopy
beta‐turn conformation in the studied material. Raman spectra of the
Basal Cornified
epithelium demonstrate that the CBP, whose relative quantity is
layer Intermediate layer layer
expressed as the sum of the beta sheet and beta‐turn conformation,
Parakeratinized +++ +++ ++
is mainly accumulated in the cornified layer. The percentage of CBP
epithelium
is greater in the cornified layer and is about 61 ± 6% (between 50%
% amount of 38 20
and 70%), while in the basal and intermediate layers, is only 45 ± 5% alpha‐keratin
(between 30% and 60%). The amount of the CBP increases to the
% amount of corneous 45 61
point to be prevalent over alpha‐keratin, as indicated in the recent beta protein
literature (Calvaresi, Eckhart, & Alibardi, 2016; Carver et al., 1990). Orthokeratinized +++ Lower Upper +
Therefore, indicating that parakeratinized epithelium undergoes epithelium (Skieresz‐ zone zone
cornification. Szewczyk et al., 2017)
++ +++
An important observation of the present research is the different
% amount of alpha‐ 43 25
coloration of the outer cells of the cornified layer of the
keratin
parakeratinized epithelium after Masson‐Goldner trichrome and
% amount of corneous 45 70
IHC staining. Topographic Masson‐Goldner staining revealed that
beta protein
the cell cytoplasm of the outermost cells of the cornified layer was
colored pale pink. The same cells, after IHC staining, showed a strong
staining reaction for alpha‐keratin. Other cells of the cornified layer
located underneath had red colored cell cytoplasm after Masson‐ lower than in the orthokeratinized epithelium. The parakeratinized
Goldner trichrome and revealed a weak staining reaction for alpha‐ epithelium covers the dorsal surface of the tongue in the domestic
keratin after IHC staining. The differences in intensity of staining of goose where food transport to the esophagus occurs (Jackowiak
the cell cytoplasm of these cells, visible after using both types of et al., 2011; Skieresz‐Szewczyk & Jackowiak, 2016). The magnitude
staining reaction, may result from the various amounts of alpha‐ of pressure on this epithelium is weaker than in places involved in
keratin and CBP. As previous IHC and Raman spectroscopy research food collection such as the mechanical papillae of the tongue
of the orthokeratinized epithelium has shown, the weak IHC staining or the lingual nail, which are covered by orthokeratinized
reaction with alpha‐keratin results from the low amount of alpha‐ epithelium. Thus, the parakeratinized epithelium does not require
keratin and high amount of CBP (Skieresz‐Szewczyk et al., 2017). In the formation of a hard and resistant cornified layer, as in the
the case of the parakeratinized epithelium, we can state that single orthokeratinized epithelium.
cells of the cornified layer are characterized by a low amount of The percentage of alpha‐keratin in the parakeratinized
alpha‐keratin and a high amount of CBP, and some have a high epithelium is similar to that in the orthokeratinized epithelium. Alpha‐
amount of alpha‐keratin and a low amount of CBP. In general, the keratin forms the intermediate filaments of the epithelial
amount of CBP in the cornified layer is higher than the amount of cells' cytoskeleton (Bragulla & Homberger, 2009). The high percentage
alpha‐keratin. of alpha‐keratin in the basal and intermediate layers of the
Raman microspectroscopy analysis reveals also the presence of parakeratinized epithelium provides a proper scaffold for epithelial
the tyrosine residues in the parakeratinized epithelium. The tyrosine cells and ensures their structural integrity. In reptiles alpha‐keratin is
and glycine‐rich regions are present in the C‐terminal domains of the present in the epidermis between scales and according to Alibardi et al.
CBP in turtles and squamates (Fraser & Parry, 2014; Gregg & Rogers, (2007) is responsible for mechanical resistance of the keratinocytes,
1986). It is believed that the physico‐chemical properties of glycine their adhesion, and ability to change shape during stretching.
and tyrosine in CBP are responsible for limited extensibility, Comparing the results of the present study in the domestic goose
microbiological resistance, and hydrophobicity, and has a protective with research in the domestic chicken, we can conclude that there
function (Alibardi, 2016; Alibardi, Toni, & Valle, 2007; Calvaresi et al., are species‐specific differences in the distribution of alpha‐keratin
2016; Fraser & Parry, 2014). and CBP in the epithelium on the dorsal surface of the tongue
Comparative analysis of the parakeratinized epithelium and (Carver & Sawyer, 1989). The differences may result from different
orthokeratinized epithelium of the tongue in the domestic goose taxonomic affiliation. Additionally, the domestic chicken uses catch
(Skieresz‐Szewczyk et al., 2017) showed a similar trend in the spatial and throw transport of food, during which the food particles are
distribution of the alpha‐keratin and CBP in the epithelial layers of directly transported from the apex to the crest of the conical papillae.
these two types of keratinized epithelia, which is shown in Table 1. The food particles do not force pressure on the epithelium of the
The percentage of CBP in the cornified layer of the paraker- body of the tongue in the chicken. Therefore the CBP occurs
atinized epithelium of the tongue in the domestic goose is about 10% occasionally in the superficial cells.
SKIERESZ‐SZEWCZYK ET AL.
The study of the cornified appendages of the epidermis, such as
scales in birds, revealed that there are only present alpha‐keratin and
CBP (Alibardi, 2004). There is no presence of the fillagrin (alpha‐helix
conformation), loricrin (alpha‐helix conformation) or transglutaminase
(alpha‐helix and beta‐sheet conformation). Wherefore the Raman
microspectroscopy analysis is a proper, useful and innovative method
to identify CBP, characterized with the beta‐sheet secondary structure
and also alpha‐keratin with the alpha‐helix structure in the epithelia of
the oral cavity in birds which undergoing cornification.
5 | CONC LU SION S
The presence of CBP in the parakeratinized epithelium and its higher
percentage in the composition of the cornified layer than in the lower
layers of the epithelium, testify that the parakeratinized epithelium,
like the orthokeratinized epithelium, undergoes cornification. At the
same time, the parakeratinized epithelium, due to the lower
percentage of CBP in the cornified layer in comparison to the
orthokeratinized epithelium and the performed feeding function, is
indicative of a weaker protective function. The distribution of alpha‐
keratin and CBP and their percentage composition in the particular
epithelial layers of the ortho‐ and parakeratinized epithelium of the
tongue in birds are probably diverse in representatives of various
feeding groups of birds, which requires further research.
A C K N O W L E D GM E N T S
The research was supported by the grant no. 507.511.62 of the Young
Researcher Program of the Faculty of Veterinary Medicine and Animal
Science Poznan University of Life Sciences, Poland financed by the
Polish Ministry of Science and Higher Education. The Raman spectro-
scopy analysis was supported by the Polish Ministry of Science and
Higher Education within Project realized at Faculty of Technical
Physics, Poznan University of Technology No. 06/65/SBAD/1952.
OR CID
Kinga Skieresz‐Szewczyk http://orcid.org/0000-0003-1031-3425
Tomasz Buchwald http://orcid.org/0000-0003-1837-402X
Mirosław Szybowicz http://orcid.org/0000-0001-8933-571X
Hanna Jackowiak http://orcid.org/0000-0001-5167-8215
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How to cite this article: Skieresz‐Szewczyk K, Buchwald T,
Szybowicz M, Jackowiak H. Alpha‐keratin and corneous beta
protein in the parakeratinized epithelium of the tongue in the
domestic goose (Anser anser f. domestica). J Exp Zool (Mol Dev
Evol). 2019;332:158–166. https://doi.org/10.1002/jez.b.22892