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Histological, histochemical, and morphometric analysis of epidermal
Leydig cells and histochemical characterization of epidermal apical cells in
juvenile and adult axolotls (Ambystoma mexicanum)
Omar Betancourt-Le´
on
a,b
, Ver´
onica Rodríguez-Mata
a
, Antonieta Martínez-Guerrero
a
,
Armando P´
erez-Torres
a,*
a
Laboratorio de Filogenia del Sistema Inmune de Piel y Mucosas, Departamento de Biología Celular y Tisular, Facultad de Medicina, Universidad Nacional Aut´
onoma de
M´
exico, Ciudad de M´
exico C.P. 04510, Mexico
b
Posgrado en Ciencias Biol´
ogicas, Unidad de Posgrado, Edicio D, 1◦Piso, Circuito de Posgrados, Ciudad Universitaria, Alcaldía Coyoac´
an, Ciudad de M´
exico C.P.
04510, Mexico
ARTICLE INFO
Keywords:
Leydig cells
Apical cells
Epidermis
Secretory cells
Paedomorphosis
ABSTRACT
Ambystoma mexicanum, also known as the axolotl, is a paedomorphic urodele. Metamorphosis can be induced
experimentally, and the most signicant changes occur in the skin. These include thinning of the epidermis,
increased keratinization of the stratied squamous epithelium, and loss of Leydig cells (LCs). Similar epidermal
changes are observed in other metamorphic urodeles. Epidermal cells are responsible for the secretory function
of the skin in juvenile amphibians, whereas dermal glands perform this function in adults after metamorphosis.
In the axolotl, this occurrence is still partially understood. The only recognized epidermal secretory cells in
juvenile A. mexicanum are the LCs, whose specic secretion products have not yet been characterized from the
histochemical standpoint. Additionally, the persistence of LCs in adulthood, when mucous and serous (granular-
protein secretion) glands are abundant, remains a matter of debate. The present study aims to describe the
morphological and histochemical changes in the epidermis of 10 cutaneous regions from juvenile (4 months old)
and adult (24 and 48 months old) non-metamorphic A. mexicanum, with a particular focus on the amount and
histochemical characteristics of LCs. Results indicate that the juvenile epidermis is a stratied cuboidal
epithelium formed by three strata: basal, spinosum (containing the LCs), and apical. The most supercial layer
contains cuboidal cells that lack the characteristics of a true stratum corneum. In adults, the stratum apical is also
formed by squamous cells, suggesting a transition to a cornied and squamous layer as age increases. Histo-
chemical methods demonstrated that LCs are most likely serous and not mucous cells. On the other hand,
cuboidal cells of the juvenile apical stratum would be responsible for producing mucous secretion components.
Morphometric analysis revealed a signicant decrease in both LCs and the epidermal thickness in the 24-month-
old adult axolotl compared to the juvenile. While LC count and epidermal thickness in the 48-month-old adult
showed a slight increase compared to the 24-month-old adult, these differences were not statistically signicant
and far lower than those observed in the juvenile axolotl, which exhibited the highest number of LCs and a
thicker epidermis. These natural axolotl epidermal changes indicate a gradual transition toward a morphology
resembling metamorphic skin as age advances. The decreased number of LCs and the transition from cuboid cells
to squamous cells in the stratum apical suggest that both cell types may naturally disappear entirely at some
point during development.
1. Introduction
Ambystoma mexicanum, also known as the axolotl, is a paedomorphic
urodele, although metamorphosis can be induced experimentally or, less
likely, occur spontaneously (Malacinski, 1978; Monaghan et al., 2014).
After induced metamorphosis, the most important changes in the skin
include thinning and increased keratinization of the epidermal stratied
squamous epithelium (Demircan et al., 2016). These ndings are similar
* Correspondence to: Edicio A, sexto piso, Facultad de Medicina, Universidad Nacional Aut´
onoma de M´
exico, Ciudad de M´
exico C.P. 04510, Mexico.
E-mail address: armandop@unam.mx (A. P´
erez-Torres).
Contents lists available at ScienceDirect
Acta Histochemica
journal homepage: www.elsevier.com/locate/acthis
https://doi.org/10.1016/j.acthis.2025.152255
Received 16 October 2024; Received in revised form 31 March 2025; Accepted 15 April 2025
Acta Histochemica 127 (2025) 152255
Available online 24 April 2025
0065-1281/© 2025 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license
( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
to those observed in metamorphic urodeles such as Hynobius retardatus
(Ohmura and Wakahara, 1998). At rst glance, the skin of amphibians
resembles that of other vertebrates, consisting of two main layers: the
dermis and the epidermis (Elkan and Cooper, 1980; Helmer and
Whiteside, 2005). The epidermis becomes more keratinized in adult
amphibians, and a stratum corneum develops (Schempp et al., 2009).
Some authors mention that the epidermis of amphibians only consists of
two strata: a basal or stratum germinative and a stratum corneum,
although some aquatic urodeles do not have a cornied epithelium
(Helmer and Whiteside, 2005; Demori et al., 2019). Other authors
recognize stratum spinosum between anurans’ basal and corneum strata
(Varga et al., 2019). The skin’s secretory function in metamorphic adults
is primarily performed by dermal glands, predominantly of two types:
mucous and serous (granular-protein secretion) (Clarke, 1997; Simmaco
et al., 1998; Wake and Koo, 2018). In contrast, the skin of juvenile
amphibians lacks both a stratum corneum and glands; instead,
epidermal cells perform secretory functions that produce mucous and
serous secretions (Quay, 1972; Quagliata et al., 2006). The secretory
epidermal cells associated with the juvenile stage of A. mexicanum are
Leydig cells (LCs), assumed to be mucous secretion cells; later, adult
axolotls develop skin mucous glands (Farkas and Monaghan, 2015). The
disappearance of LCs has been reported in the natural metamorphosis of
the Hynobius retardatus species (Ohmura and Wakahara, 1998). How-
ever, these cells persist during development in the arrested meta-
morphosis of H. retardatus (Ohmura and Wakahara, 1998). In contrast to
A. mexicanum, other studies on LCs in species such as Ambystoma opa-
cum, Ambystoma tigrinum, and Taricha torosa employ specimens in the
larval or juvenile phase since those are the stages of development in
which LCs are present (Hay, 1961; Kelly, 1966; Hui and Smith, 1976).
The process by which dermal glands replace the cellular secretory ele-
ments is well-dened in metamorphic amphibians. However, it is
important to clarify that the paedomorphic condition of A. mexicanum
maintains the LCs in the epidermis during the adult stage (Gerling et al.,
2012), which makes their role during development even more
intriguing. Additionally, no other epidermal secretory cells have been
reported. This unique characteristic of the A. mexicanum epidermis may
represent a distinctive feature at the cellular and tissue levels.
The main argument for postulating that LCs produce mucus com-
ponents is the fact that they are periodic acid-Schiff (PAS)-positive,
which demonstrates molecules with monosaccharide residues contain-
ing "neutral" hexose sugars such as glucose, galactose, mannose, and
fucose—the principal sugars present in polysaccharides, proteoglycans,
and glycoproteins (Kiernan, 2010). However, this presumed functional
property of LCs has not been reliably proved in juvenile and adult
A. mexicanum. Gerling et al. (2012) closely analyzed the number, dis-
tribution, and development of these cells in larvae up to 100 days after
hatching. During this period of development, a linear increment in LCs
was observed as body size increased. Besides, after 6 months of age, fully
differentiated LCs in all body regions were crowded with toluidine
blue-positive granules, which was also observed in the LCs of an 8-year--
old axolotl. The present study aimed to analyze the histological char-
acteristics of the epidermis in 10 cutaneous regions from juvenile (4
months) and adult (24 and 48 months) non-metamorphic A. mexicanum,
focusing on the number, distribution, and histochemistry of LCs. He-
matoxylin and eosin (H&E) staining and various histochemical tech-
niques were employed, including periodic acid-Schiff (PAS), alcian blue
pH 2.5 (AB), Giemsa (G), Masson’s trichrome (MT), and colloidal iron
(CI). Three epidermal strata were identied: basal, spinosum (contain-
ing LCs), and apical. The stratum apical transitions from cuboidal cells in
juveniles to increasingly squamous cells in adults, suggesting a pro-
gression toward a true stratum corneum with age. Histochemical anal-
ysis indicates LCs are serous rather than mucous cells, with mucous
secretion likely produced by cuboidal cells of the stratum apical pre-
dominant in juvenile axolotl. LC density and epidermal thickness
signicantly decreased from juvenile to 24-month adults, with minimal
change between 24- and 48-month specimens. These changes suggest
gradual development toward metamorphic skin morphology, with both
LCs and apical cells potentially disappearing completely during
development.
2. Materials methods
2.1. Laboratory animals
Female wild-type specimens of A. mexicanum species (n =3) aged 4,
24, and 48 months were used in this study. The sex of the specimens did
not inuence our skin examination, and the 4-month-old specimen’s sex
was indeterminate. The axolotls were acquired from a wildlife man-
agement property (PIMVS, Predios o Instalaciones queue Manejan Vida
Silvestre per its Spanish acronym) registered as SEMARNAT-PIMVS-CR-
157-MEX/19 "The Paradise of the Axolotls" in Mexico City, Mexico. In
the laboratory, specimens were housed individually in 20 L tanks lled
with dechlorinated tap water and equipped with air pumps and acti-
vated carbon lters. Water changes of 30 % volume were conducted
periodically. Throughout their housing, various physicochemical pa-
rameters, such as temperature, water oxygen saturation, lighting, and
pH, were monitored to ensure optimal conditions for the laboratory
animals. The axolotls were fed three times a week with a diverse diet
that included guppy sh, tubifex worms, water eas, brine shrimp, and
occasionally Tropical® "Axolotl sticks" dry food. All procedures for
handling, care, feeding, and skin sampling adhered to the Guide for the
Care and Use of Laboratory Animals (8th edition) published by the
National Research Council of the National Academies of the United
States. Additionally, the project was approved by the Institutional
Committee for the Care of Laboratory Animals (CICUAL, per its Spanish
acronym) and the Research and Bioethics Committees of the Faculty of
Medicine at the National Autonomous University of Mexico (UNAM),
with approval number "FM/DI/106/2023". During their captivity in the
laboratory, no spontaneous mortality or illness occurred among the
specimens, and they demonstrated good adaptation to their diet and
environment overall.
2.2. Euthanasia and skin sampling
Specimens were anesthetized by immersion in 0.5 % tricaine meth-
anesulfonate (MS-222, Sigma-Aldrich, E10521) diluted in water tanks
for 10 minutes at room temperature. After conrming that the axolotls
showed no motor activity or reactivity to manipulation, they were
immersed in Bouin xative for 30 minutes to complete euthanasia.
Subsequently, skin samples of 0.5 cm² were taken from the following
body regions (Fig. 1): dorsal skin of the head (DSH), ventral skin of the
head (VSH), dorsal skin of the trunk at the forelimb level (DST/F),
ventral skin of the trunk at the forelimb level (VST/F), right intercostal
fold (RIF), left intercostal fold (LIF), dorsal skin of the trunk at the
hindlimb level (DST/H), ventral skin of the trunk at the hindlimb level
(VST/H), caudal ridge skin (CRS), and ventral skin of the tail (VST).
Fixation of the skin samples continued by immersion in Bouin xative
for an additional 90 minutes at room temperature. The remainder of the
specimens were xed in neutral buffered 10 % formaldehyde. Before
xation, a ventral medial incision was made from the mandible to the
cloaca to expose the internal organs to the xative solution, thereby
preserving and optimizing the use of the axolotls for other research
purposes.
2.3. Processing skin samples for histological studies
After xation, the skin samples were washed under running water
until the excess xative was removed, indicated by the absence of yel-
lowing in the wash water. The samples were then dehydrated using a
series of increasing ethanol concentrations: 60 %, 70 %, and 80 % for
1 hour each, followed by two successive 30-minute changes in 96 % and
100 % ethanol. The samples underwent two successive 20-minute
O. Betancourt-Le´
on et al.
Acta Histochemica 127 (2025) 152255
2
xylene treatments to remove the ethanol completely. Inltration was
performed using two changes of histological parafn at 56◦C, the rst
for 90 minutes and the second for 1 hour. The skin samples were then
embedded in parafn to obtain 4
μ
m thick slices. These slices were
stained with Hematoxylin and Eosin (H&E, Hematoxylin, Merck, Art.
4305. Colour Index Number: 75290. Eosin Y disodium salt. Sigma-
Aldrich, E4382. Colour Index Number: 45380) which identies acidic
or anionic structures (basophilic) by staining blue-magenta, as well as
basic components (acidophilic), generally proteins, which stain pinkish
orange. H&E staining was performed according to the Hematoxylin and
Eosin procedure of the AFIP manual of Prophet et al. (1994).
2.4. Quantication and distribution analysis of Leydig cells (LCs) in
A. mexicanum epidermis
The LCs were counted in non-serial, H&E-stained histological skin
sections spaced 100
μ
m apart to prevent recounting. Quantitative
analysis was performed using a BX50 Olympus microscope equipped
with a digital camera and the Innity Analyze software (v6.3.0),
employing a 10X objective calibrated with a micrometer ruler. LC
quantication was conducted over a total epidermal area of 5 mm² for
each cutaneous region, measured in
μ
m² using the same method. LC
density (cells/mm²) of all body regions was calculated for each sample
by dividing the number of LCs by the corresponding epidermal area
measured in mm². A total number of samples per age and cutaneous
region was obtained. These results were represented with a graph of the
Empirical Cumulative Distribution Function (ECDF) (Fig. 3). To detect
differences in the distributions, the non-parametric Mann–Whitney U
and Kolmogorov–Smirnov tests were used (Fig. 3).
2.5. Regional distribution analysis of Leydig cells (LCs), epidermal
thickness areas, and radar plots
The distribution of LCs number and epidermal thickness areas across
different axolotl ages (4, 24, and 48 months) for each sampled cutaneous
region was analyzed using either the average or median value for each
region-age combination, depending on data normality. Normality was
assessed using the Shapiro-Wilk test (Supplementary Tables 1 and 2).
Radar plots were designed to represent these results (Figs. 4 and 5). To
determine the signicance of observed changes and account for non-
normal distributions, the non-parametric Kruskal-Wallis test was
employed to analyze differences in each triad of ages by region. Pairwise
differences were identied using a post-hoc Dunn’s test with Bonferroni
correction. The analysis code is available in the following repository: htt
ps://github.com/OmarsBetancourts/Leydig-axolotls
2.6. Histochemical analysis of Leydig cells (LCs) and other epidermal
cells in A. mexicanum
All skin samples were processed to obtain parafn tissue sections, as
mentioned above. The following histotechnological methods were per-
formed according to the manual AFIP of Prophet et al. (1994): Periodic
acid-Schiff (PAS) AFIP modication to detect neutral polysaccharides,
staining purple. The dye used was basic fuchsin, Sigma-Aldrich, 215597.
Colour Index Number: 42500. Alcian blue (AB) at pH 2.5 was used to
identify acidic, sulfate, or carboxylate polysaccharides and pro-
teoglycans, which stained light blue. The dye used was Alcian blue 8GX,
Sigma-Aldrich, A5268. Colour Index Number: 74240. Masson trichrome
(MT) highlights collagen bers and cationic and anionic proteins in blue
and red, respectively. The dyes used were Biebrich scarlet, MP Bio-
medicals, IC15485525. Colour Index Number: 26905. Acid fuchsin,
Sigma-Aldrich, F8129, Colour Index Number: 42685. Aniline blue,
Biognost, CAB-P-25G, Colour Index Number: 42780. The colloidal iron
(CI) technique was used to evaluate mucins, mucin-like molecules, and
acidic mucosubstances (including polysaccharides, proteoglycans, and
glycoproteins), which dyed dark blue. The Giemsa stain (G) technique
was used to analyze the orthochromatic and metachromatic properties
of cytoplasmic granules. Although this technique was based on the
protocol of Prophet et al. (1994), individual dyes were replaced by a
composite dye, Giemsa´s Azur-eosin-Methylene blue, Merck, Art. 9203.
Additionally, Nuclear fast red, Sigma-Aldrich, 229113, Colour Index
Number: 60760, was used as a nuclei counterstaining dye in the AB and
CI techniques.
3. Results
3.1. Animal welfare in the laboratory
As described in the Materials and Methods section, the specimens did
not exhibit behavior indicative of stress, such as hyperactivity, buoyancy
issues, or surface oxygen uptake (oropharyngeal breathing). Further-
more, the specimens responded well to the diet, including non-living
food items.
3.2. Euthanasia and skin sampling
The specimens were anesthetized through immersion, and after
approximately 10 minutes, they were removed from the anesthetic so-
lution to assess their state of deep anesthesia using tactile stimuli. When
no motor responses were observed following the stimuli, euthanasia was
performed by immersing the specimens in a xative. Subsequently, skin
samples were collected and immersed in the xative solution. The
collection and processing of samples were conducted without signicant
difculties.
3.3. Processing skin samples for histological studies
3.3.1. Epidermal structure and histological characteristics of Leydig cells
(LCs) in A. mexicanum at 4 months of age
The epidermis of the 4-month-old juvenile axolotl (Fig. 2) is a
stratied cuboidal epithelium with three strata: 1) stratum basale,
formed by a layer of pyramidal cells with clear cytoplasm and pleo-
morphic nuclei; 2) stratum spinosum or intermediate layer, distin-
guished by the presence of LCs located at different levels, forming 3 or 4
Fig. 1. Diagram of A. mexicanum showing the body regions of skin sampling.
Corporal regions. 1) Dorsal skin of the head (DSH), 2) Ventral skin of the head
(VSH), 3) Dorsal skin of the trunk at the forelimb level (DST/F), 4) Ventral skin
of the trunk at the forelimb level (VST/F), 5) Right intercostal fold (RIF), 6)
Left intercostal fold (LIF), 7) Dorsal skin of the trunk at the hindlimb level
(DST/H), 8) Ventral skin of the trunk at the hindlimb level (VST/H), 9) Caudal
ridge skin (CRS) and 10) Ventral skin of the tail (VST).
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Acta Histochemica 127 (2025) 152255
3
(caption on next page)
O. Betancourt-Le´
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Acta Histochemica 127 (2025) 152255
4
layers in the DSH, VSH, DST/F, and VST/F regions (as well as DST/H,
VST/H, RIF, LIF; see supplementary gure 1), but only one layer in the
CRS and VST regions. These cells, the largest in the epidermis, exhibit
central, spherical, or ovoid nuclei with irregular proles and abundant
euchromatin. Their cytoplasm is strongly eosinophilic and diffuse,
containing clear vesicles (some with central eosinophilic staining) in the
VSH, DST/F, and VST/F regions. In the DSH region, the cytoplasm is less
eosinophilic than in the aforementioned regions, but most vesicles have
eosinophilic central granular staining with a clear surrounding halo. The
LCs on the outermost stratum spinosum layer display morphological
polarity, with a more eosinophilic apical or supercial domain
frequently containing vesicles with compact, strongly eosinophilic
(acidophilic) granular centers. Their basal cytoplasmic domain pre-
dominantly contains clear, unstained vesicles. In both the CRS and VST
regions, LC cytoplasm is entirely occupied by clear vesicles. The stratum
spinosum also comprises small clear cells with pleomorphic nuclei
located among the LCs in all analyzed regions. Melanocytes are identi-
able in the dorsal region skin; 3) the stratum apical, the uppermost
epidermal layer, is well-differentiated and consists of 2–4 layers of
eosinophilic cells. The deep cells are at, while the supercial cells are
cuboidal, characterized by a basophilic covering with a occulant and
scalloped appearance. A distinct basement membrane separates the
epidermis from the dermis. LCs appear to rest directly on the basement
membrane in the CRS region.
3.3.2. Epidermal structure and histological characteristics of Leydig cells
(LCs) in A. mexicanum at 24 months of age
The epidermis of the 24-month-old adult axolotl (Fig. 2) predomi-
nantly exhibited stratied squamous epithelium at the DSH, VST/F, and
CRS regions (as well as DST/H, VST/H, RIF, and LIF; see Supplementary
Figure 1), while stratied cuboidal epithelium was present at the VSH,
DST/F, and VST regions. Both types of epithelia displayed the same
strata described in the 4-month-old axolotl. In some skin regions, a
combination of cuboidal and at cells was observed in the outermost
layer of the stratum apical. LCs, present in all cutaneous regions and
organized in one to three sublayers within the stratum spinosum,
exhibited a remarkable load of intensely eosinophilic cytoplasmic
granules. These granules were distributed perinuclearly but separated
from the nuclei by a zone of cytoplasm containing few or no granules. An
eosinophilic Langerhans network, readily observable in all LCs,
appeared to contain all cytoplasmic granules and separate them from the
cell membrane. Most LCs displayed a clear polarized distribution of
cytoplasmic granules towards the stratum apical, with some of the most
supercial ones separated from the external medium by a single layer of
at cells. A basophilic covering with a occulant and scalloped
appearance was observed on the epidermal surface in all analyzed skin
regions. A stratum basale composed of pleomorphic cells (spindle-sha-
ped, cuboidal, or cylindrical, depending on the skin region) separated
the LCs from the basement membrane, although it was not uncommon to
observe these cells attached to the basement membrane. The melano-
cytes and small clear cells with pleomorphic nuclei, located among the
LCs, showed no differences from those described in the skin of the 4-
month-old axolotl.
3.3.3. Epidermal structure and histological characteristics of Leydig cells
(LCs) in A. mexicanum at 48 months of age
The epidermis of the 48-month-old adult axolotl (Fig. 2) exhibited
predominantly stratied squamous epithelium at the DSH and DST/F
regions (as well as DST/H, VST/H, RIF, and LIF; see Supplementary
Figure 1), whereas stratied cuboidal epithelium was observed at the
VSH and VST regions. The VST/F and CRS regions displayed both
cuboidal and squamous cells in the supercial layer of the stratum
apical. Several notable changes were identied in the LCs across all skin
regions. In the DSH, VSH, DST/F, VST/F (Fig. 2), DST/H, VST/H, RIF,
Fig. 2. Leydig cells and stratum apical of A. mexicanum (H&E) H&E stain. Dorsal skin of the head (DSH), Ventral skin of the head (VSH), Dorsal skin of the trunk at
the forelimb level (DST/F), Ventral skin of the trunk at the forelimb level (VST/F), Caudal ridge skin (CRS) and Ventral skin of the tail (VST). The scale bar in the
photomicrographs represents 25 µm. The age of each specimen is indicated in each column. In the 4-month-old axolotl, the DSH region contains LCs with some
eosinophilic cytoplasmic granules (yellow arrows), while the VSH, DST/F, and VST/F regions show LCs with eosinophilic cytoplasm but no granules (circles). The
CRS and VST regions lack these staining patterns. A basophilic layer is observed over the stratum apical in the DSH, VSH, DST/F, and VST regions (black arrows). At
24 months, LCs exhibit abundant eosinophilic cytoplasmic granules (yellow arrows), tending towards apical morphological polarization. In the 48-month-old
axolotl, some LCs display eosinophilic granules (yellow arrows), but most contain numerous basophilic granules (red arrows). Other LCs maintain the eosino-
philic cytoplasmic appearance observed in the 4-month-old specimen (circles). Notably, the cutaneous VST region lacks LCs. All epidermal strata contain elongated
or rounded cells with clear, granule-free cytoplasm.
Fig. 3. Age-related changes in Leydig Cells (LCs) density in the axolotl´s epidermis. Fig. 3. Empirical cumulative distribution function (ECDF) illustrating the Leydig
cell (LCs) density per mm² across different ages (4, 24, and 48 months). The inset (red box) provides a magnied view near the median density, emphasizing clear,
age-dependent shifts. Older groups display a leftward displacement, indicating reduced LC density. Statistical analysis revealed signicant differences between age
groups: Mann–Whitney U test showed signicant differences between 4 and 48 months (p =5.86 ×10⁻¹⁸) and between 24 and 48 months (p =1.57 ×10⁻¹³), but no
signicant difference between 4 and 24 months (p =0.577). Kolmogorov–Smirnov tests also unveiled substantial differences in distribution shape among all age
comparisons: 4 vs. 24 months (p =4.19 ×10⁻⁷), 4 vs. 48 months (p =5.05 ×10⁻¹⁴), and 24 vs. 48 months (p =1.24 ×10⁻¹⁵).
O. Betancourt-Le´
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Acta Histochemica 127 (2025) 152255
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and LIF regions (see Supplementary Figure 1), the LCs closest to the
stratum basale, and some in an intermediate position within the stratum
spinosum, exhibited characteristics similar to those described in the
VSH, DST/F, VST/F (Fig. 2), VST/H, RIF, and LIF regions (see Supple-
mentary Figure 1) of the 4-month-old axolotl. These LCs displayed
strongly eosinophilic and diffuse cytoplasm with clear vesicles, a few of
which contained central eosinophilic staining. However, the remaining
LCs possessed intensely basophilic cytoplasmic granules, while others,
particularly those near the apical layer, exhibited both cytoplasmic
features. Notably, the most surprising change was the absence of LCs in
the VST region, where the stratum spinosum comprised rounded clear
cells and few melanocytes. A occulant and scalloped basophilic
covering on the epidermal surface was observed in all cutaneous re-
gions, primarily where the supercial layer of the stratum apical
retained its cuboidal shape (VSH and VST, as well as VST/F and CRS
regions).
3.4. Quantication and distribution analysis of Leydig cells (LCs) in
A. mexicanum epidermis
To characterize age-related changes in Leydig cell (LCs) density,
empirical cumulative distribution functions (ECDFs) of LC density
(cells/mm²) were generated for each age group (4, 24, and 48 months;
Fig. 3). A total of 5 mm
2
of epidermal thickness was measured for each
cutaneous region for each axolotl studied. Nonparametric statistical
analyses were selected, as these methods are robust to deviations from
normality and small samples. Specically, Mann–Whitney U tests were
employed to assess pairwise differences in median LC densities, while
Kolmogorov–Smirnov tests were utilized to compare the shapes of the
distributions.
The ECDF clearly illustrates an age-dependent shift in LC density
(Fig. 3). Although the Mann–Whitney U test revealed no statistically
signicant difference between the 4- and 24-month groups (p =0.577),
a discernible decreasing trend in median LC density was observed, from
3.08 cells/mm² at 4 months to 2.98 cells/mm² at 24 months (Table I).
This decline became statistically signicant at 48 months, with a pro-
nounced reduction in median LC density to 2.76 cells/mm², signicantly
lower compared to both the 4-month (p =5.86 ×10⁻¹⁸) and 24-month
groups (p =1.57 ×10⁻¹³).
Further analysis using the Kolmogorov–Smirnov test conrmed sig-
nicant differences in the distribution shapes across all age compari-
sons: 4 vs. 24 months (p =4.19 ×10⁻⁷), 4 vs. 48 months (p =5.05 ×
10⁻¹⁴), and 24 vs. 48 months (p =1.24 ×10⁻¹⁵). Notably, the LC density
distribution at 48 months exhibited clear bimodality, characterized by
two distinct peaks—one near zero and another around 3 cells/
mm²—accompanied by increased variability (SD =1.14, IQR =1.12 at
48 months), relative to younger groups (SD =0.80; IQR =0.79 at 4
months). These observations emphasize the emergence of substantial
epidermal heterogeneity with advancing age.
Descriptive statistics further reinforce these ndings (Table I). At 4
months, the LC density distribution was strongly negatively skewed
(-1.27) and exhibited high kurtosis (5.36), indicating a concentration of
data points at higher LC densities typical of the juvenile epidermis. At 24
months, skewness became slightly positive (0.25), and kurtosis signi-
cantly decreased to 3.26, reecting greater data dispersion and struc-
tural diversication associated with maturation. At 48 months,
skewness reverted to negative (-0.99), and kurtosis further decreased to
3.19, consistent with the observed bimodal distribution, indicative of
enhanced variability and increased epidermal complexity in older
specimens.
3.5. Regional distribution analysis of Leydig cells (LCs), epidermal
thickness areas, and radar plots
The comparison among various cutaneous regions in juvenile (4
Fig. 4. Central tendency values of Leydig cells (LCs) count across different
regions and ages. Number of LCs per cutaneous region. Dorsal skin of the head
(DSH), Ventral skin of the head (VSH), Dorsal skin of the trunk at the forelimb
level (DST/F), Ventral skin of the trunk at the forelimb level (VST/F), Right
intercostal fold (RIF), Left intercostal fold (LIF), Dorsal skin of the trunk at the
hindlimb level (DST/H), Ventral skin of the trunk at the hindlimb level (VST/
H), Caudal ridge skin (CRS) and Ventral skin of the tail (VST). The radar plot
illustrates the distribution of LC counts across various cutaneous regions
(5 mm² of epidermis for each region) in A. mexicanum at three different ages: 4
months (red), 24 months (green), and 48 months (blue). The average cell count
was utilized for each cutaneous region when the sample distribution was
normal; otherwise, the median cell count was employed. The results of the
normality tests are available in Supplementary Table I. The gure shows a
pronounced and statistically signicant decrease in LC numbers with advancing
age across nearly all analyzed cutaneous regions. This age-related decline in LCs
becomes apparent when comparing the values at 4 months, 24 months, and
48 months.
Fig. 5. Central tendency values of epidermal areas across different regions and
ages. Epidermal area or thickness by body region. Dorsal skin of the head
(DSH), Ventral skin of the head (VSH), Dorsal skin of the trunk at the forelimb
level (DST/F), Ventral skin of the trunk at the forelimb level (VST/F), Right
intercostal fold (RIF), Left intercostal fold (LIF), Dorsal skin of the trunk at the
hindlimb level (DST/H), Ventral skin of the trunk at the hindlimb level (VST/
H), Caudal ridge skin (CRS) and Ventral skin of the tail (VST). The radar plot
illustrates the variations in epidermal thickness areas (µm²) across different
body regions and ages in axolotls. The green line, representing the 24-month-
old adult, consistently falls within the boundaries of the red (4-month-old ju-
venile) and blue lines (48-month-old adult). This gure demonstrates that
epidermal thickness signicantly and consistently decreases with age. These
observations, clearly depicted in the radar plot, support the inverse relationship
between age and both LCs presence and epidermal thickness in axolotls. The
data highlights the substantial inuence of aging on the cellular and structural
composition of axolotl skin.
O. Betancourt-Le´
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Acta Histochemica 127 (2025) 152255
6
months old) and adult (24 and 48 months old) A. mexicanum revealed
signicant differences in LC numbers, as demonstrated by both the radar
plot (Fig. 4) and the statistical comparisons (Table II). The radar plot
shows fewer LCs in nearly all cutaneous regions of the 24-month-old
specimen compared to the 48-month-old specimen. However, the LC
numbers in the 48-month-old specimen never exceeded those observed
in the 4-month-old specimen, except for the CRS region. As indicated in
Table II, at least one signicant difference was present in each region.
The results conrm signicant decreases in LC numbers for almost all
pairs except for the CRS (4 vs. 24 months), LIF (24 vs. 48 months), and
VST/F (24 vs. 48 months and 4 vs. 48 months) regions. This visualiza-
tion provides a clear comparative view of the differences in LC numbers
across individual cutaneous regions and complements the global
changes in LC density described previously.
The epidermal thickness analysis across various cutaneous regions of
A. mexicanum axolotls aged 4, 24, and 48 months revealed signicant
differences, as demonstrated by the radar plot (Fig. 5) and the statistical
comparisons presented in Table III. At 4 months, epidermal thickness
areas are relatively larger in most regions compared to those of adult
axolotls. At 24 months, the epidermal thickness area showed a marked
reduction in most regions. In the 48-month-old specimen, the epidermal
thickness areas generally increased compared to the 24-month-old
specimen but remained lower than those of the 4-month-old specimen
in several regions. Regions such as VST/F, VST/H, DST/F, DST/H, LIF,
and RIF exhibited signicantly larger epidermal thickness areas in ju-
venile axolotl. This visualization suggests a trend where the epidermal
thickness areas notably decrease from the juvenile stage to the 24-
month-old adult, then increase slightly in the 48-month-old adult
specimen, but do not return to the values observed in the 4-month-old
juvenile specimen.
3.6. Histochemical analysis of Leydig cells (LCs) in the epidermis of
A. mexicanum
The histochemical characteristics of juvenile and adult A. mexicanum
LCs were examined using PAS, AB pH 2.5, G, MT, and CI staining
techniques. H&E staining (Fig. 2) revealed that the cytoplasmic granules
of LCs become distinctly acidophilic only in the skin of the DSH region of
the 4-month-old axolotls. By 24 months of age, the LC granules are
strongly acidophilic in all skin regions. At 48 months, the LC granules
exhibit a dye shift towards basophilia, and in the VST region, the LCs
with their distinctive granules disappear. Histochemically, only LC
granules from the DSH region of the 4-month-old axolotl were PAS-
positive and surrounded by a clear halo (Fig. 6). The cytoplasm of LCs
in the VSH region (which also exhibits polarization towards the stratum
apical), DST/F, and VST/F regions were also PAS-positive, while the LCs
of the CRS and VST regions were PAS-negative. As previously noted,
granules of LCs from 24- and 48-month-old axolotls were acidophilic
and basophilic, respectively. However, both types of granules were PAS-
positive, while the epidermis of the VST region lacked PAS-positive cells.
Another noteworthy observation was that the supercial layer of the
stratum apical was PAS-positive in the DSH, VSH, and DST/F cutaneous
regions. In contrast, in the other cutaneous regions, including the VST
region that lacks typical LCs, the positivity was localized toward the
outer surface of the supercial layer, presenting a scalloped appearance
(Fig. 6).
Histochemistry with AB, pH 2.5 (Fig. 7), revealed that LC granules
were not positive in the ten skin regions analyzed from the three axolotls
studied. The cytoplasm of the LCs appeared pink or pale pink; however,
some cytoplasmic granules were strongly stained red by the nuclear fast
red used to counterstain the epidermal cell nuclei. Another striking
nding was the AB-positivity exhibited by the scalloped outer surface of
the epidermis in all cutaneous regions studied, with greater intensity
observed in the DSH, VSH, and VST/F regions across all three axolotls
ages. The 24-month-old axolotl displayed the lowest intensity of AB
staining in all regions, while the lowest positivity in the 48-month-old
axolotl was observed in the DST/F and CRS regions. In the juvenile
axolotl, AB positivity was noted in the apical cytoplasm of the cuboidal
cell layer of the stratum apical in the VSH, DST/F, and VST/F regions, as
well as in underlying layers, except for the CRS and VST regions.
The Giemsa staining (Fig. 8) revealed that the cytoplasmic granules
of LCs are intensely acidophilic (eosinophilic), exhibiting a regional and
age distribution similar to that observed with H&E staining. This pattern
was evident in the DSH region of the 4-month-old axolotl and all skin
regions of 24- and 48-month-old adults, except for the VST region of the
48-month-old adult axolotl. Notably, the epidermal strata were highly
basophilic in the 4-month-old juvenile axolotl. This dye afnity
decreased in the 24-month-old adult axolotl and shifted completely to
strong acidophilia in the 48-month-old adult axolotl. Additionally, the
scalloped outer surface of the epidermis displayed metachromatic
staining in all cutaneous regions of the juvenile axolotl, with a more
pronounced stain in the DSH, CRS, and VST regions. In contrast, this
metachromatic staining was incipient only in the DSH region of the 24-
month-old adult axolotl and absent in all other analyzed skin regions of
both 24- and 48-month-old adults.
Staining with the MT method (Fig. 9) selectively revealed a basement
membrane with regional variations in thickness and aniline blue stain
intensity. Additionally, stratum apical cells in 24- and 48-month-old
axolotls exhibited blue cytoplasmic staining, particularly in the super-
cial squamous cells. A well-developed blue Langerhans network was
observed in LCs, most prominently in the 24-month-old adult axolotl,
while a less distinct and greenish gray one appeared in the 48-month-old
specimen. This network forms a compartment between the cytoplasmic
periphery and the central perinuclear region, known as the hofplasma,
hofbereich, or court plasma, which contains cytoplasmic granules. These
LCs and epidermal features were absent in the juvenile axolotl. Notably,
MT staining demonstrated heterogeneity in LC granule properties. In the
juvenile axolotl, only the LCs in the DSH, VSH, and VST/F regions dis-
played dark granules surrounded by light halos. In the 24-month-old
axolotl, most LC granules across all skin regions stained dark brown,
with occasional reddish granules. The greatest staining heterogeneity
was observed in the 48-month-old adult axolotl, even within individual
LCs. In the DSH, VSH, and CRS regions, granules stained emerald-green,
while in the DST/F and VST/F regions, most LC granules appeared
reddish, with some emerald-green. The CRS region LCs best exemplied
the presence of these two granule types. MT staining also conrmed the
absence of LCs in the VST region of this axolotl.
Histochemistry with CI (Fig. 10) revealed intense positivity,
Table I
Age-dependent changes in Leydig cell density in the epidermis of Ambystoma mexicanum.
Age Mean Median SD IQR Skewness Kurtosis
4 months 3.000001 3.082428 0.801527 0.791124 −1.26728 5.35839
24 months 3.061149 2.98158 1.232604 1.553302 0.247116 3.256049
48 months 2.44068 2.756921 1.141175 1.122958 −0.9919 3.194792
Descriptive statistics of Leydig cell (LCs) density (cells/mm²). The table summarizes the mean, median, standard deviation (SD), interquartile range (IQR),
skewness, and kurtosis values. The data demonstrates a progressive decline in median LC density from juvenile (4 months) to older adult (48 months) axolotls. Notably,
LC density at 48 months shows a marked decrease in mean and median, accompanied by increased variability (higher SD and IQR) compared to the youngest age group
(4 months), suggesting increased epidermal heterogeneity associated with aging in A. mexicanum.
O. Betancourt-Le´
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Acta Histochemica 127 (2025) 152255
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Table II
Comparative analysis of the number of LCs in A. mexicanum across ages and
different cutaneous regions using Kruskal-Wallis and Dunn’s post-hoc tests.
Region Kruskal-
Wallis Test
(p-value)
Group
1
Group
2
Dunn’s test
(adjusted p-
value)
Signicance
Levels
DSH 2.15E−23 24
months
24
months
4
months
4
months
48
months
48
months
3.68 ×10
−22
4.94 ×10
−10
0.0004
****
****
***
VSH 4.97E−24 24
months
24
months
4
months
4
months
48
months
48
months
1.33 ×10
−22
2.73 ×10
−09
0.001
****
****
**
DST/F 5.10E−18 24
months
24
months
4
months
4
months
48
months
48
months
1.45 ×10
−18
0.008
1.96 ×10
−09
****
**
****
VST/F 0.000365 24
months
24
months
4
months
4
months
48
months
48
months
0.0002
0.226
0.14
***
Ns
Ns
RIF 2.45E−17 24
months
24
months
4
months
4
months
48
months
48
months
9.98 ×10
−18
0.0001
8.11 ×10
−06
****
***
****
LIF 5.25E−16 24
months
24
months
4
months
4
months
48
months
48
months
8.55 ×10
−14
1
9.12 ×10
−14
****
Ns
****
DST/
H
3.75E−25 24
months
24
months
4
months
4
months
48
months
48
months
8.52 ×10
−25
1.22 ×10
−07
5.23 ×10
−06
****
****
****
VST/
H
1.24E−24 24
months
24
months
4
months
4
months
48
months
48
months
2.98 ×10
−10
2.64 ×10
−06
3.51 ×10
−25
****
****
****
CRS 1.06E−20 24
months
24
months
4
months
4
months
48
months
48
months
0.42
5.78 ×10
−20
1.95 ×10
−13
Ns
****
****
VST 6.90E−37 24
months
24
months
4
months
4
months
48
months
48
months
0.036
2.70 ×10
−24
1.55 ×10
−32
*
****
****
Results of the Kruskal-Wallis test and post-hoc Dunn’s test with Bonferroni
correction for multiple comparisons across different cutaneous regions and
axolotl ages (4 months, 24 months, and 48 months). The table presents Kruskal-
Wallis’s test p-values, age comparisons (specimen 1 and specimen 2), adjusted p-
values, and signicance levels. Signicant p-values (p<0.05) indicate signi-
cant differences between the age groups in the respective cutaneous regions.
Signicance levels are indicated as follows: ns (not signicant), *p<0.05,
**p<0.01, ***p<0.001, and ****p<0.0001. The results highlight signicant
differences in the number of Leydig cells between most ages compared across
various cutaneous regions.
Table III
Comparative analysis of epidermal thickness areas in A. mexicanum across
different ages and cutaneous regions using Kruskal-Wallis and Dunn’s post-hoc
tests.
Region Kruskal-
Wallis Test
(p-value)
Group
1
Group
2
Dunn’s test
(adjusted p-
value)
Signicance
Levels
DSH 3.02E−31 24
months
24
months
4
months
4
months
48
months
48
months
2.09 ×10
−30
2.97 ×10
−11
6.92 ×10
−07
****
****
****
VSH 5.43E−28 24
months
24
months
4
months
4
months
48
months
48
months
5.74 ×10
−21
1.01 ×10
−17
1
****
****
Ns
DST/F 4.19E−24 24
months
24
months
4
months
4
months
48
months
48
months
1.85 ×10
−24
2.53 ×10
−06
7.86 ×10
−09
****
****
****
VST/F 8.20E−24 24
months
24
months
4
months
4
months
48
months
48
months
1.80 ×10
−20
2.14 ×10
−13
0.2253329
****
****
Ns
RIF 1.77E−27 24
months
24
months
4
months
4
months
48
months
48
months
7.00 ×10
−27
1.45 ×10
−09
3.92 ×10
−06
****
****
****
LIF 2.08E−26 24
months
24
months
4
months
4
months
48
months
48
months
1.64 ×10
−24
4.11 ×10
−11
0.00018345
****
****
***
DST/
H
6.69E−32 24
months
24
months
4
months
4
months
48
months
48
months
3.38 ×10
−29
1.39 ×10
−13
0.0002387
****
****
***
VST/
H
2.18E−19 24
months
24
months
4
months
4
months
48
months
48
months
2.12 ×10
−17
1
3.46 ×10
−16
****
Ns
****
CRS 1.14E−34 24
months
24
months
4
months
4
months
48
months
48
months
2.35 ×10
−15
1.10 ×10
−32
3.52 ×10
−05
****
****
***
VST 4.12E−22 24
months
24
months
4
months
4
months
48
months
48
months
4.69 ×10
−22
1.35 ×10
−08
0.0003279
****
****
***
Results of Kruskal-Wallis test and Dunn’s post-hoc test for multiple comparisons
of epidermal thickness in axolotls at three ages. The cutaneous regions analyzed
include CRS, DSH, DST/F, DST/H, LIF, RIF, VSH, VST, VST/F, and VST/H. For
each region, the p-values of the Kruskal-Wallis test are provided, along with the
age comparisons (Group 1 vs. Group 2), the adjusted p-values from Dunn’s test,
and the signicance levels. The signicance levels are denoted as follows: ns (not
signicant), * (p<0.05), ** (p<0.01), *** (p<0.001), and **** (p<0.0001).
O. Betancourt-Le´
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O. Betancourt-Le´
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Acta Histochemica 127 (2025) 152255
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represented by blue staining, in the stratum apical and on the external
surface of the epidermis across various skin regions of axolotls at
different ages. In the 4-month-old axolotl, this intense staining was
observed in the DSH, VSH, DST/F, VST/F, CRS, and VST regions. The 24-
month-old axolotl was prominent in the DSH, VSH, VST, and DST/F
regions. All skin regions of the 48-month-old axolotl exhibited this
intense blue staining. Moderate staining intensity was noted in the VST/
F region of the juvenile axolotl and the CRS region of the 24-month-old
adult. The LC granules displayed a red stain, likely due to the nuclear
fast red counterstaining dye, in specic regions: the DSH and VSH of the
juvenile axolotl and the DSH and DST/F of both adult axolotls. Addi-
tionally, in the 48-month-old adult axolotl, the cytoplasmic granules of
LCs in the VSH, VST/F, and CRS regions showed intense red staining.
The remaining skin regions examined did not exhibit granule staining
with nuclear fast red. All the histochemical properties of the LCs are
summarized in Table IV.
3.6.1. Histochemistry of the stratum apical
As described above, the H&E stain revealed that the stratum apical of
the epidermis, whose surface cells dene the epithelium type, exhibited
histological changes corresponding to the axolotl’s age. In the juvenile
axolotl, the supercial cells displayed a cuboidal shape. In the 24-
month-old adult axolotl, these cells were squamous in many skin re-
gions, with some regions retaining cuboidal morphology. In the 48-
month-old axolotl, the squamous appearance of the surface apical cells
predominated, although some regions of the ventral skin contained islets
of cuboidal cells adjacent to squamous cells. A basophilic covering with
a occulant and scalloped appearance was observed at the supercial
domain of the cells and on the epidermis surface in all skin regions, both
in juvenile and adult axolotls (Fig. 2). Notably, the cytoplasm and
occulating material of the basophilic body surface were stained with
PAS, AB pH 2.5, and CI in all three axolotls studied (Figs. 6, 7, 10, and
Table V). Giemsa staining demonstrated a metachromatic pattern of this
occulant material in all cutaneous regions of the juvenile axolotl and
the DSH region of the 24-month-old axolotl (Fig. 8). No distinctive
staining pattern in this epidermal area was observed using the MT
method. see supplementary gures 6, 7, 8, 9 and 10 for histochemical
results of the skin regions RIF, LIF, DST/H and VST/H.
4. Discussion
During amphibian metamorphosis, frogs, toads, and salamanders
undergo gradual changes in the structure and function of their organs
and tissues as they transition from larva to tadpole to adult. These
changes include tail shortening and absorption, lung development,
growth of legs, and dietary and skin adaptations that enable them to live
in both aquatic and terrestrial environments. However, some Anura and
salamander (urodele) species remain fully aquatic, with few or no
morphofunctional changes for terrestrial life. Ambystoma mexicanum, a
species of urodele, is emblematic of paedomorphosis, characterized by
the retention of gills and an aquatic lifestyle throughout their life
(Shaffer, 1993; Voss et al., 2009). Although morphological meta-
morphosis does not occur in A. mexicanum, it has been suggested that
cryptic metamorphosis does occur at the tissue and molecular level
(Tompkins and Townsend, 1977; Wakahara, 1996). An example of this
phenomenon is the presence of LCs, a characteristic component of uro-
dele larval skin, which is typically lost by adulthood in metamorphic
salamanders such as Lissotriton italicus and Hynobius retardatus (Ohmura
and Wakahara, 1998; Perrotta et al., 2012). Interestingly, the histolog-
ical changes of the skin in natural and experimentally induced meta-
morphosis are similar to those observed in metamorphic amphibian
species, including depletion or total loss of LCs (Demircan et al., 2016).
During the larval stage of A. mexicanum, LCs are the only identied and
recognized secretory components of the skin, characterized by the
presence of distinctive cytoplasmic granules. However, the permanence
of these cells throughout all stages of the axolotl development, as well as
the composition of their secretion and denitive function, have not been
reliably elucidated (Gerling et al., 2012; Brunelli et al., 2022). Also,
larval and juvenile axolotls lack skin glands that only develop in
adulthood. In the present study, we conducted histological,
non-enzymatic histochemical, and morphometric analyses of the
epidermis of juvenile (4 months old) and adults (24 and 48 months of
age) A. mexicanum to investigate potential tissue changes associated
with developmental progression, focusing on the characteristics and
number of LCs, as well as in the epidermal strata.
4.1. With increasing age, the axolotl’s stratum apical transitions from
cuboidal to squamous morphology, with a decrease in Leydig cell (LC)
count
The epidermis of the analyzed skin regions in juvenile and adult
axolotls comprised three strata: basal, spinosum (or intermediate), and
apical. Our observations revealed that the apical and spinous strata
exhibited the most signicant changes. The supercial layer of the
stratum apical transformed from cuboidal (in the juvenile) to squamous
(in adults) cell morphology. Concurrently, the stratum spinosum dis-
played variations in the presence, reduction, and even absence of LCs.
These structural modications likely contributed to changes in the
epidermal thickness, with the juvenile possessing a thicker epidermis
than adults. Furthermore, these alterations were associated with distinct
histochemical and functional characteristics of both strata in the juve-
nile and adult axolotls.
A noteworthy nding is the cephalocaudal trend in the decrease and
absence of LCs. While the juvenile axolotl exhibited more LCs and adult
axolotls generally showed a decrease in LC numbers, the stratum spi-
nosum of the VST region displayed LCs only in the 24-month-old adult
axolotl. Although we lack an explanation for this phenomenon, it sug-
gests that the tail may not require the secretory function of LCs during
the juvenile stage and at 48 months of age. Beyond the number of LCs in
each skin region, the histofunctional features at different axolotl ages
are also relevant. In this context, the LCs described by Gerling et al.
(2012) in 100-day-old axolotls still possessed numerous unstained
cytoplasmic vacuoles (vesicles), which could be interpreted as a state of
functional immaturity or incomplete differentiation. In our 4-month-old
juvenile axolotl, LC granules maturation appeared to have progressed, as
some LCs exhibited stained granules (and cytoplasm) arranged with
apical polarization, suggesting the acquisition of secretory function
despite the continued presence of unstained vesicles. Similarly, our
Fig. 6. Leydig cells (LCs) and stratum apical of A. mexicanum (PAS). Legends and Description. PAS stain. Dorsal skin of the head (DSH), Ventral skin of the head
(VSH), Dorsal skin of the trunk at the forelimb level (DST/F), Ventral skin of the trunk at the forelimb level (VST/F), Caudal ridge skin (CRS) and Ventral skin of the
tail (VST). The scale bar in the photomicrographs represents 25 µm. The age of each specimen is indicated in each column. In the 4-month-old specimen, PAS
positivity (light to intense purple-magenta color) is prominent in the apical domain of the cuboidal cells of the stratum apical, as well as in the occulating material
covering the outer surface (black arrows). This characteristic is particularly noticeable in the DSH and VSH regions. The LCs display PAS-positive cytoplasm
(circles), which is more intense in the apical portion. The LCs exhibit unstained vesicles, some with PAS-positive centers or compact PAS-positive granules, more
evident in the DSH and VSH regions (yellow arrows). In the 24-month-old specimen, the stratum apical contains PAS-positive cuboidal and squamous-shaped cells.
The PAS-positive occulating material on the outer surface is more pronounced in the DSH and VSH regions (black arrows). The LCs exhibit abundant, well-
differentiated, and strongly PAS-positive granules (yellow arrows). In the 48-month-old specimen, some basal LCs resemble those of the 4-month-old specimen
(circles). At the same time, LCs in the spinous stratum also exhibit abundant and strongly PAS-positive granules similar to those in the 24-month-old axolotl
(yellow arrows).
O. Betancourt-Le´
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Acta Histochemica 127 (2025) 152255
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Fig. 7. Leydig cells (LCs) and stratum apical of A. mexicanum (AB) Legends and Description. AB pH 2.5 Stain. Dorsal skin of the head (DSH), Ventral skin of the head
(VSH), Dorsal skin of the trunk at the forelimb level (DST/F), Ventral skin of the trunk at the forelimb level (VST/F), Caudal ridge skin (CRS) and Ventral skin of the
tail (VST). The scale bar in the photomicrographs corresponds to 25 µm. The age of each specimen is indicated in each column. In the 4-month-old specimen, intense
AB pH 2.5-positive staining was observed in the apical domains (black arrows) and a few cytoplasmic granules of cuboidal-shaped cells from thestratum apical
Notably, an AB-positive occulating material covered all analyzed cutaneous regions. In 24- and 48-month-old adult axolotls, cuboidal cells and more squamous-
shaped cells, along with the occulating material cover (black arrows), displayed AB pH 2.5-positive staining, predominantly in ventral cutaneous regions. LC
granules were not AB pH 2.5 positive; however, in some cutaneous regions, the cytoplasmic granules exhibited a strong red stain (yellow arrows).
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24-month-old axolotl presented LCs with granules of diverse staining
properties, representing a higher stage of morphofunctional differenti-
ation than that described by Gerling et al. (2012) in a 1-year-old axolotl.
The LCs of the 48-month-old specimen seemed to be undergoing a
renewal process (although no signicant mitotic events were observed).
Basal LCs showed poorly differentiated granules and cytoplasm, while
those in the stratum spinosum, particularly those in apical positions,
presented well-differentiated granules with varying staining properties.
This suggests that this adult axolotl possessed LCs at different stages of
maturation, some resembling those of the juvenile axolotl, with staining
properties distinct from those of the 24-month-old axolotl. Notably, the
granules exhibited remarkable basophilia with H&E staining, although
the granules were acidophilic when stained with Giemsa at both ages.
The proposed interpretation to understand the aforementioned
changes is based on the process of metamorphosis. This process involves
a radical transformation in an organism’s morphology and function,
coinciding with or followed by a change in its life history. Meta-
morphosis occurs in biphasic organisms with larval and adult stages
adapted to different lifestyles. The distinction between larval and ju-
venile stages is not always clear. While there is no universally accepted
denition of a larva, a common morphological criterion denes it as "a
morphological state eliminated by the metamorphic transition to the
juvenile" (Callery, 1999). During metamorphosis, de novo organogenesis
occurs, and pre-existing organs undergo large-scale remodeling,
including the lungs, liver, eye, brain, spinal cord, nose, pituitary, and
blood. Notable changes also occur in the immune, musculoskeletal, and
integumentary systems (Brown and Cai, 2007). The larval amphibian
skin comprises numerous cell types with specic functional and topo-
graphic relationships and distinct differentiation and longevity periods.
As development progresses, skin complexity increases in cellular
composition. However, some epidermal cell types that originate early in
larval life disappear before the onset of prometamorphosis, while others
persist throughout larval and adult life, presumably maintaining the
same function (Fox, 1981). Generally, larval urodeles possess LCs, while
metamorphic adults have multicellular dermal glands (Smirnov, 2003).
Recent research has recognized that the neotenic pedomorphosis con-
dition of A. mexicanum and other urodele species results in LCs persisting
throughout development (Gerling et al., 2012; Demircan et al., 2016;
Brunelli et al., 2022). Although morphological metamorphosis does not
occur, cryptic metamorphosis involving cellular and biochemical
changes is recognized (Tompkins and Townsend, 1977; Wakahara,
1996). We attribute the observed morphological and likely functional
changes in specimens of three different ages to this cryptic meta-
morphosis. We suggest that the skin of A. mexicanum may be tran-
sitioning at the tissue level towards a metamorphic skin, although
further study of environmental and endocrine factors modulating this
ontogenically prolonged process is necessary. Some early metamorphic
events directed by thyroid hormones are known to occur in Ambystoma
despite its paedomorphic condition (Callery, 1999). Moreover, indi-
vidual cell types comprising an organ can be direct targets of thyroid
hormones, generating autonomous programs of independent cells
(Brown and Cai, 2007). This could explain the presence of "islands" of
cuboidal epidermal cells in the stratum apical of adult specimens, as well
as the presumed renewal or dedifferentiation of some basal LCs in the
48-month-old specimen, which resembles those of the juvenile axolotl.
4.2. Epidermal thickness and Leydig cells (LCs) count are reduced in the
adult stage
Due to the small sample size of axolotls studied, we utilized the most
representative parameter analyzed from the distributions of epidermal
thickness area measurements. The radar graph illustrates that the 4-
month-old juvenile axolotl generally exhibited the thickest epidermis
across the 10 skin regions examined. In contrast, the 24-month-old adult
axolotl showed a considerable decrease in epidermal thickness
compared to the juvenile specimen. The 48-month-old adult axolotl had
more epidermal thickness than the 24-month-old adult, but it was still
considerably thinner compared to the 4-month-old juvenile. These
ndings contrast with those of Gerling et al. (2012), who reported that
throughout the lifespan of A. mexicanum, "the epidermis of the head is
slightly thicker than in the trunk and notably thicker than in the tail; in
the trunk, the ventral epidermis is thicker than on the lateral surface and
the thinner epidermis is located in the tail" (head >trunk >tail). In our
4-month-old specimen, we observed that the trunk regions (DST/F,
VST/F, RIF, LIF, DST/H, and VST/H) tended to be slightly thicker than
those of the head (DSH and VSH) but noticeably thicker than those of the
tail (head <trunk >tail) (Fig. 5). The 24-month-old specimen exhibited
a relatively uniform epidermal thick area across all body regions (head
≈trunk ≈tail), while the 48-month-old specimen displayed a pattern of
epidermal area variation similar to that of the 4-month-old axolotl,
albeit with lower values.
The analysis of LC distribution revealed that adult axolotls possess
fewer of these cells than their juvenile counterparts, with the 24-month-
old adult axolotl exhibiting the lowest number of LCs. Across all three
axolotl ages, the tail consistently displayed the lowest concentration of
LCs compared to other skin regions. This observation suggests that the
tail primarily serves to locomotion and that its cutaneous secretory
function is transient. Despite methodological differences between the
study by Gerling et al. (2012) and the present research, such as the use of
semi-thin skin sections versus histological parafn sections, variations in
histological staining techniques, and differences in sample selection,
both studies conrm decreased LCs along the cephalocaudal axis.
Furthermore, they both demonstrate a reduction in the LC number from
the larval stage (up to 80 days post-hatch) to the juvenile stage (4
months of age) and subsequently to adulthood (24 and 48 months of
age).
The reduction of LCs, coupled with the thinning of the epidermis and
the transformation of supercial cells of the stratum apical from
cuboidal to squamous morphology as age progresses, may be interpreted
as ontogenetic changes related to a process of cryptic metamorphosis.
These changes observed in the present study are associated with the
gradual formation of dermal glands from epidermal crests, suggesting an
apparent switch from the secretory function of LCs, which predominates
in larval and juvenile stages of the axolotl, to newly developed serous
and mucous glands in adult organisms. Considering this information and
presuming that the tail of A. mexicanum serves primarily as a locomotion
rather than a secretory structure, it becomes clear why the epidermis in
this body region consistently appears as the thinnest and contains the
fewest LCs in the specimens analyzed.
Fig. 8. Leydig cells (LCs) and stratum apical of A. mexicanum (G) Legends and Description. G stain. Dorsal skin of the head (DSH), Ventral skin of the head (VSH),
Dorsal skin of the trunk at the forelimb level (DST/F), Ventral skin of the trunk at the forelimb level (VST/F), Caudal ridge skin (CRS) and Ventral skin of the tail
(VST). The scale bar in the photomicrographs represents 25 µm. The age of each specimen is indicated in each column. In the 4-month-old specimen, most epidermal
cells exhibited basophilia, while LCs displayed acidophilic or eosinophilic cytoplasm (pink-red color) (circles). Some LCs in the DSH region contained eosinophilic
granules surrounded by a clear halo (yellow arrows). Epidermal basophilia decreased in all cutaneous regions of the 24-month-old adult axolotl, but the LC granules
remained intensely acidophilic (yellow arrows). Notably, the epidermis of the 48-month-old adult axolotl was highly acidophilic, as were the granules of the LCs
(yellow arrows). A metachromatic occulating material (purple color) (black arrows) covering the epidermal surface was prominent in the 4-month-old specimen,
becoming fainter in the 24-month-old adult axolotl (DSH) and markedly eosinophilic in the 48-month-old adult axolotl.
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Acta Histochemica 127 (2025) 152255
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Fig. 9. Leydig cells (LCs) and stratum apical of A. mexicanum (MT) Legends and Description: MT Stain. Dorsal skin of the head (DSH), Ventral skin of the head (VSH),
Dorsal skin of the trunk at the forelimb level (DST/F), Ventral skin of the trunk at the forelimb level (VST/F), Caudal ridge skin (CRS) and Ventral skin of the tail
(VST). The scale bar in the photomicrographs represents 25 µm. The age of each specimen is indicated in each column. In the 4-month-old axolotl, the epidermis
stained reddish-ochre, while the LCs appeared gray with few black granules (circles). The 24-month-old axolotl exhibited a bluish coloration in the epidermis, with
LCs displaying a blue-tinged Langerhans network on the cytoplasmic periphery (yellow arrows), surrounding numerous dark-red-brown granules (red arrows). In
some skin regions of the 48-month-old axolotl, the LCs contained emerald-green colored granules (black arrows) (DSH, VSH), while others had dark-red-brown
granules (red arrows) (VST/F, DST/F), and some regions showed both types of granules (CRS). The basal membranes were well-dened and blue stained.
O. Betancourt-Le´
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Acta Histochemica 127 (2025) 152255
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Fig. 10. Leydig cells (LCs) and Legends and description. CI stain. Dorsal skin of the head (DSH), Ventral skin of the head (VSH), Dorsal skin of the trunk at the
forelimb level (DST/F), Ventral skin of the trunk at the forelimb level (VST/F), Caudal ridge skin (CRS) and Ventral skin of the tail (VST). The bar of the photo-
micrographs corresponds to 25 µm. The age of each specimen is indicated in each column. The stratum apical in the 4-month-old axolotl exhibits a cuboidal
morphology and displays intense positive staining with CI (black arrows). As axolotls mature to 24 and 48 months, this stratum transitions to a squamous
morphology while maintaining CI positivity in the apical domain, appearing as a occulent material on the epidermal surface (black arrows). Some cuboidal cells in
adult axolotls retain CI positivity like that observed in the 4-month-old specimen. The LCs and their granules were CI-negative; however, they demonstrated a strong
afnity for nuclear fast red staining, as observed with AB pH 2.5.
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Acta Histochemica 127 (2025) 152255
15
4.3. Granules of Leydig cells (LCs) contain basic glycoproteins, not
mucus; the stratum apical potentially synthesizes epidermal mucus
secretion
Since their discovery in the epidermis of salamander larvae, LCs were
initially identied as mucous cells (Leydig, 1853, "Schleimzelle").
Extensive histological studies of LCs have proposed structural support
network functions, holocrine but also exocrine secretion mechanisms,
and mucus production for protection purposes (Hay, 1961; Kelly, 1966;
Andrew and Hickman, 1974; Kato and Kurihara, 1987; Jarial, 1989;
Toyoshima and Shimamura, 1992). However, their precise function re-
mains unknown. While the notion that LCs secrete mucus persists and
appears true for L. italicus (Brunelli et al., 2022), our results indicate that
this is not the case for A. mexicanum at any of the three ages studied.
Both the AB technique, used to identify acidic mucopolysaccharides or
glycosaminoglycans (GAGs) containing sulfated or carboxylated groups,
and the CI technique, employed to evaluate mucins, mucin-like mole-
cules, and acidic mucosubstances, failed to produce the characteristic
dark blue coloration (Prophet et al., 1994) in LC secretory granules.
Furthermore, the PAS-positivity and acidophilia, along with the nega-
tive reaction to AB and CI, suggest that some components of the LC
granules are basic glycoproteins rather than mucins. Mucins are typi-
cally negatively charged acid glycoproteins due to their high proportion
of sialic acid and glucuronic acid residues (Kiernan, 2010). These nd-
ings pave the way for biochemical and functional studies of putative
basic glycoproteins, hypothesizing that they may play a role in the
innate immune response of A. mexicanum against viral and bacterial
adhesion and invasion of the cutaneous surface, as Andrew and Hickman
(1974) suggested for the LCs secretion function.
According to the staining results (H&E and Giemsa techniques) and
non-enzymatic histochemistry (AB and CI), cells in the stratum apical of
axolotls are responsible for mucous secretion. This was observed in both
the 4-month-old juvenile axolotl, which lacked dermal mucous glands,
and the adults, where this epidermal mucous secretion complements the
dermal mucous glands. The supercial cuboidal and squamous cells, and
some deeper cells in this stratum, consistently exhibited basophilia,
metachromasia, PAS positivity, and dark blue coloration indicative of
AB and CI technique positivity (Prophet et al., 1994) in their apical
domain. These characteristics were often present on the external surface
Table IV
Staining and histochemistry results for LCs.
Staining/
histochemistry
result for LCs
4 months 24 months 48 months
H&ECytoplasm and
some immature
acidophilic
granules.
Cytoplasm and
mature
acidophilic
granules.
Cytoplasm and
granules were
mostly acidophilic.
Some cells with
basophilic granules
are present
PAS Cytoplasm and
some immature
granules were
weakly positive.
Cytoplasm and
mature granules
were intensely
positive.
Cytoplasm and
mature granules
were intensely
positive.
AB pH 2.5 Cytoplasm and
granules were
not stained.
Cytoplasm and
granules were not
stained. Nuclear
fast red stained
granules.
Cytoplasm and
granules were not
stained. Nuclear
fast red stained
granules.
GCytoplasm and
some immature
acidophilic
granules.
Cytoplasm and
mature
acidophilic
granules.
Cytoplasm and
mature acidophilic
granules.
MT Cytoplasm and
granules were
not stained.
Langerhans
network aniline
blue positive and
reddish-brown
dark granules.
Langerhans
network stained
light blue, and
reddish and
emerald-green
stained granules.
CI Cytoplasm and
granules were
not stained.
Cytoplasm and
granules were not
stained. Nuclear
fast red stained
granules.
Cytoplasm and
granules were not
stained. Nuclear
fast red stained
granules.
Table V
Histology and histochemistry of the stratum apical.
Staining/
histochemistry
result for LCs
4 months 24 months 48 months
H&ECuboidal shape,
acidophilic
cytoplasm with a
basophilic apical
domain. Cover with
basophilic
occulating
material.
Cuboidal shape, but
more squamous-
shaped cells with
acidophilic
cytoplasm. Cover
with basophilic
occulating
material.
Mostly
squamous-
shaped cells
with “islands” of
cuboidal cells.
Cover with
basophilic
occulating
material.
PAS Cuboidal shape
with some
cytoplasmic
granules and apical
domain strongly
PAS-positive. Cover
with PAS-positive
occulating
material.
Cuboidal shape, but
more squamous-
shaped cells
covered with PAS-
positive
occulating
material.
Mostly
squamous-
shaped cells
with “islands” of
cuboidal cells,
both PAS-
positives. Cover
of PAS-positive
occulating
material.
AB pH 2.5 Cuboidal shape
with some
cytoplasmic
granules at apical
domains strongly
AB- positive. Cover
with AB-positive
occulating
material.
Cuboidal shape, but
more squamous-
shaped cells with
AB-positive
occulating
material.
Mostly
squamous-
shaped cells
with “islands” of
cuboidal cells,
both AB-
positives. Cover
of AB-positive
occulating
material, mainly
in skin ventral
regions
GCuboidal shape
with some
metachromatic
cytoplasmic
granules at the
apical domains.
Cover of
metachromatic
occulating
material.
Cuboidal shape, but
more squamous-
shaped cells with
weakly or absent
metachromatic
occulating
material.
Mostly
squamous-
shaped cells
with “islands” of
cuboidal cells,
both became
acidophilic, as
well as the cover
of occulating
material.
MT Cuboidal shape
with reddish
staining of the
cytoplasm. Cover of
non-stained
occulating
material.
Cuboidal shape but
more squamous-
shaped cells with
faint blue or gray
staining. Cover of
non-stained
occulating
material.
Mostly
squamous-
shaped cells
with “islands” of
cuboidal cells,
with apical
domains and
faint blue or
gray staining.
Cover of non-
stained
occulating
material.
CI Cuboidal shape
with the apical
domains and the
cover occulating
material, both
strongly AB-
positives.
Cuboidal shape, but
more squamous-
shaped cells with
apical domains, and
cover of
occulating both
strongly AB-
positives.
Mostly
squamous-
shaped cells
with “islands” of
cuboidal cells,
with apical
domains, and
cover of
occulating
material both
strongly AB-
positives.
O. Betancourt-Le´
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Acta Histochemica 127 (2025) 152255
16
as ne cytoplasmic granules. Such tinctorial properties suggest the
presence of acidic mucopolysaccharides or glycosaminoglycans with
sulfated or carboxylated groups, simple neutral and sulfated poly-
saccharides, as well as carboxylated acid glycoproteins such as mucins
and other acidic mucosubstances—all of which are constituent compo-
nents of mucous secretion (Largen and Woodley, 2008; Heiss et al.,
2009; von Byern et al., 2015; Pereira et al., 2018). To our knowledge,
these results are the rst to demonstrate that cuboidal cells of the stra-
tum apical are the primary site of mucus production in juvenile axolotls
and adults before developing dermal mucous glands.
This histofunctional scenario identied in the epidermis of
A. mexicanum aligns partly with the concept of secretory epidermal cell
dichotomy typical of larval amphibians and other anamniote organisms
(Quay, 1972). There is a consensus that this dichotomy is partly struc-
tural, consisting of two cell types: spherical or goblet mucocytes with
basal nuclei and pale cytoplasm that stain histochemically for acidic and
sulfated glycosaminoglycans and a heterogeneous group of
protein-granular secretion cells with varying morphological, functional,
and histochemical characteristics depending on the species. The primary
common feature in this dichotomy is storing secretion products in
discrete entities or cytoplasmic granules (Quagliata et al., 2006). This
hypothesis of secretory cell dichotomy holds true in the epidermis of
juvenile axolotls and during early adulthood, before the development of
dermal glands. However, the changes observed with advanced age in the
studied axolotls deviate from the aforementioned hypothesis, empha-
sizing instead the emergence of a potential and gradual process of
cryptic metamorphosis.
4.4. Additional staining properties of Leydig cells (LCs) with nuclear fast
red
The staining properties of a 0.1 % solution of (NFR) with 5 %
aluminum sulfate are frequently used for counterstaining dark pink to
reddish chromatin of cell nuclei. In the absence of aluminum salt, a
slightly acidied solution of NFR acts as an ordinary anionic dye that can
be used instead of eosin Y after hemalum (alum–hematoxylin) nuclear
stain; consequently, cytoplasm and extracellular structures (collagen
bers) are colored faint pink (Frank et al., 2007). In the present study,
NFR was used to stain the nuclei of epidermal cells after AB-pH2.5 and
CI histochemistry. Surprisingly, in addition to staining the usual red
nuclei using Al mordant, the LC granules also showed a strong red stain.
The mechanism of nuclear staining by NFR has not been investigated, so
an explanation for this nding is not available. As mentioned above,
NFR without a mordant is an anionic dye that does not stain nuclei dark
pink to red but does stain the cytoplasm light pink. It is likely that NFR in
solution with aluminum sulfate, which is electrically neutral, interacts
with Al³ ⁺ cations, changing their tinctorial behavior and favoring the
staining of the anionic components of chromatin such as DNA, RNA, and
some negatively charged non-histone acidic proteins (due to the pres-
ence of glutamic acid and aspartic acid). Following this reasoning,
NFR-Al₂(SO₄)₃ would be staining anionic, acidic, non-mucin proteins
since LC granules were not staining with AB pH 2.5 and CI. These pu-
tative acid proteins, along with the putative basic glycoproteins, ac-
cording to H&E, PAS, and Giemsa staining results, support the
hypothesis that the LCs secretion could be relevant in the innate immune
response of A. mexicanum.
The orthochromatic dye afnity of the LC granules for Biebrich
scarlet (BS) in adult axolotls, particularly in 24-month-old organisms,
suggests the presence of basic proteins, given the acidic pH context of
the MT staining method (Prophet et al., 1994). BS is an acid azo dye used
as a cytoplasmic stain in MT in combination with acid fuchsin (Cooksey,
2020). Aniline blue, a basic dye with a positive charge, exhibits a high
tinctorial afnity for collagen and negatively charged acidic proteins
under acidic conditions, such as those in the MT method.
The LC granules in 48-month-old axolotls were BS-positive. Inter-
estingly, some LCs simultaneously presented emerald-green granules, as
observed in the CRS cutaneous region. Additionally, some LCs contained
only emerald-green granules. A plausible interpretation for this histo-
chemical reaction, based on the dyeing properties of BS and aniline blue,
is that the LCs of 48-month-old axolotls synthesize a new type of acidic
protein stainable with aniline blue, which in some LCs coexists with
basic proteins that have an afnity for BS. This nding suggests that
there is potential for further histochemical studies of LCs using BS in
different controlled pH solutions (ranging from acidic to basic) to
identify specic amino acid residues associated with these putative basic
proteins and to better understand the aniline blue staining results
described above (Phifer et al., 1973).
5. Conclusions
The epidermis of Ambystoma mexicanum exhibits histological, his-
tochemical, and morphometric changes during the gradual transition
from juvenile to adult stage, which can be interpreted as a cryptic
metamorphosis in a neotenic paedomorphic organism. As axolotls age,
the epidermal thickness decreases, and the epithelium transforms from
juvenile to adult form, with the stratum apical shifting from cuboidal to
squamous shape. A general trend of decreased Leydig cells in the
cephalocaudal direction is observed as axolotls mature, with these cells
disappearing entirely in the ventral tail skin of adult axolotls. Although
abundant mitotic events were not observed, the histochemical charac-
teristics of Leydig cells and their granules indicate changes in differen-
tiation and maintenance of cell turnover during the analyzed
developmental stages. Leydig cells in axolotls should be classied as
serous granular cells, secreting basic and acidic glycoproteins, with full
differentiation completed in the adult stage. In the juvenile stage,
cuboidal cells of the stratum apical are responsible for producing mu-
cous secretion components when the dermal mucous glands character-
istic of the adult axolotl skin have not yet developed.
This research contributes to the expanding knowledge of skin biology
and provides insights into the functional role of LCs in A. mexicanum, a
paedomorphic urodele amphibian. While endemic to Lake Xochimilco,
Mexico, this species has been worldwide distributed due to its signi-
cance as an animal model in biological, biotechnological, and biomed-
ical studies of vertebrates.
Funding
Support from the Research Division of the Faculty of Medicine of
UNAM (project registration: "FM/DI/106/2023").
CRediT authorship contribution statement
Betancourt-Le´
on O: Conceptualization, Methodology, Investigation,
Formal analysis, Writing original draft, and Writing review & editing.
Rodríguez-Mata V: Investigation, Methodology. Martínez-Guerrero A:
Data curation, Formal analysis. P´
erez-Torres A: Conceptualization,
Methodology, Formal analysis, writing original draft and writing review
& editing, Funding acquisition.
Declaration of Generative AI and AI-assisted technologies in the
writing process
Authors declared that the content of manuscript was not reviewed
and edited with IA assistant and are full responsible to the content of the
published article.
Acknowledgments
To the graduate program in biological sciences at the National
Autonomous University of Mexico (UNAM) since this work was carried
out as part of the requirements for obtaining the doctorate in sciences
degree of the rst author.
O. Betancourt-Le´
on et al.
Acta Histochemica 127 (2025) 152255
17
To the Secretary of Science, Humanities, Technology and Innovation
(SECIHTI) of Mexico for its support.
Declarations of interests
On behalf of all co-authors, I declare and assure, as the corresponding
author, that there is no conict of interest.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.acthis.2025.152255.
Data Availability
We have shared the link to my data/code at the attached le step
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