Lipopolysaccharide (LPS) is a component of the Gram-negative bacterial cell wall and is considered a potent inflammatory inducer. Empagliflozin (EMPA) is a sodium-glucose co-transporter 2 inhibitor used in treatment of type 2 diabetes mellitus. Interleukin-6/Janus kinase/signal transducer and activator of transcription 3 (IL-6/JAK/STAT3) signaling pathway is essential in driving inflammation, fibrosis, and epithelial–mesenchymal transition (EMT). Tracheal basal stem cells are responsible for epithelial regeneration after injury. This study aimed to investigate the potential protective effects of EMPA against LPS-induced tracheal injury, with emphasis on EMT and tracheal basal stem cells. Sixty adult male rats were allocated into four groups: control; EMPA, which received EMPA (10 mg/kg/day) via oral gavage for 28 days; LPS, which received single intratracheal dose of LPS (5 mg/kg) on day 1; and LPS + EMPA, which received single LPS dose and EMPA for 28 days. Tracheal specimens were processed for biochemical, histological, immunohistochemical and RT-quantitative real-time PCR analysis. Compared to the LPS group, the LPS + EMPA group showed marked improvement of histological, ultrastructural ,and biochemical alterations including maintenance of epithelial and cilia integrity, decreased lipid peroxidation, stimulated antioxidant enzyme activities, reduced serum inflammatory cytokines, increased percentage area of E-cadherin (an epithelial marker) immunostaining, decreased percentage area of fibrosis, vimentin (a mesenchymal marker), and pro-apoptotic Bax; and enhanced percentage area of cytokeratin 5/6 (CK5/6) (stem cell marker). Moreover, EMPA downregulated the IL-6/JAK/STAT3 pathway involved in EMT. In conclusion, EMPA alleviated LPS-induced tracheal injury through its antioxidant and anti-inflammatory properties, downregulation of IL-6/JAK/STAT3 signaling pathway, and preservation of the tracheal basal stem cells.
DNA methylation is considered a well-regulated mechanism involved in cellular differentiation, particularly in the differentiation of mesenchymal stem cells into chondrocytes. TET family proteins potentially regulate demethylation by the oxidative conversion of 5-methylcytosine (5-mC) into hydroxymethyl cytosine (5-hmC) and then into formylcytosine (5-fC) and carboxyl cytosine (5-caC) in a series of reactions. However, the precise role of TET proteins and the impact of 5-hmC dynamics during chondrocyte differentiation remain unclear. This study aimed to investigate the potential association of 5-hydroxymethylation with TET family proteins and human telomerase reverse transcriptase (hTERT) gene expression during the differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes. Methodology involves hMSCs differentiation into chondrocytes over 16 days of culture, with successful differentiation confirmed by Alcian Blue staining and chondrogenic marker gene (ACAN and COL2A1). Gene and protein expressions of TET1, TET2, TET3, and hTERT were analyzed through real-time polymerase chain reaction (RT-PCR) and western blot. Global changes in DNA 5-hmC level quantified using dot blot and enzyme-linked immunosorbent assay (ELISA). Locus-specific changes within the hTERT promoter gene were analyzed through locus-specific PCR. We observed a substantial increase in the expression levels of TET family proteins TET1 and TET2, as well as high global 5-hmC levels during chondrocyte differentiation. However, human telomerase reverse transcriptase (hTERT) expression was reduced both at the messenger RNA (mRNA) and protein levels throughout the differentiation process. Furthermore, epigenetic analysis revealed that elevated 5-hmC enrichment at the hTERT promoter region was strongly associated with the downregulation of hTERT expression. Our findings suggest that 5-hmC may serve as a potential epigenetic biomarker and regulatory element in chondrocyte differentiation, offering new insights into skeletal development and cartilage-related disorders.
Excessive sucrose consumption is associated with various metabolic disorders, including obesity, diabetes, and liver dysfunction. Although oxidative stress is a well-established mechanism contributing to these conditions, its effects on matrix metalloproteinase (MMP) activity in the liver remain poorly understood. This study evaluated the impact of a high-sucrose diet on oxidative stress, MMP-2 and MMP-9 activities, insulin signaling and collagen deposition in the liver of Swiss mice. Male Swiss mice (protocol no. 7,422,050,723) were divided into 2 groups with 16 animals each—control and high-sucrose diet—and were fed for 4 weeks. Liver samples were collected for histomorphological analysis of steatosis, inflammatory infiltrates, and collagen deposition, as well as for biochemical evaluation of antioxidant enzyme activity, oxidative stress markers, MMP activity, and AKT phosphorylated at serine 473. The high-sucrose diet induced metabolic imbalance, steatosis, and inflammatory infiltration, accompanied by oxidative stress in the liver, evidenced by reduced catalase activity (CAT) and glutathione levels (GSH), along with increased protein carbonyls (PCO) and thiobarbituric acid reactive substances (TBARS) levels. Additionally, MMP-9 activity and collagen deposition were higher in mice fed the high-sucrose diet. Notably, AKT phosphorylation at Ser-473 residue was reduced, indicating impaired insulin signaling. These findings suggest that a high-sucrose diet induces hepatic oxidative stress, disrupts insulin signaling, and promotes MMP-9 activation, potentially contributing to liver histopathological changes and dysfunction.
Pulmonary hypertension (PH) is a progressive disease characterized by pulmonary vascular remodeling and increasing right ventricular (RV) afterload. Extracellular matrix (ECM) remodeling is central to PH, but its temporal dynamics across lung and RV tissue remains insufficiently defined. Extra domain A-containing fibronectin (ED-A+ Fn) is a fetal fibronectin splice variant re-expressed during tissue injury and chronic inflammation. This study investigated temporal and spatial ED-A+ Fn expression in monocrotaline (MCT)-induced PH in rats. Male Sprague–Dawley rats were analyzed at day 7, 14, 21, and 28 after MCT injection. Disease progression was assessed by echocardiography, right heart catheterization, histology, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and serum enzyme-linked immunosorbent assay (ELISA). MCT induced PH with increasing RV systolic pressure, RV dilatation, reduced tricuspid annular plane systolic excursion (TAPSE), and worsening pulmonary and myocardial tissue damage. ED-A+ Fn was nearly absent in controls but increased progressively at the protein level in lung and RV tissue. Significant upregulation was detected from day 14 onward in the lung and at later stages in the RV. ED-A+ Fn was spatially associated with macrophage accumulation and remodeling markers, including α-smooth muscle actin and tenascin-C. By contrast, ED-A+ Fn messenger RNA (mRNA) levels did not change significantly. Circulating ED-A+ Fn was increased in PH animals and correlated with impaired RV function, higher RV systolic pressure, and pulmonary and RV myocardial histopathological damage. ED-A+ Fn is progressively re-expressed during experimental PH and reflects cardiopulmonary remodeling in tissue and circulation. These findings support ED-A+ Fn as a dynamic biomarker of disease progression and a potential translational target in PH.
The preBötzinger complex (preBötC) is a medullary network that generates the inspiratory phase of respiratory rhythm in mammals and depends critically on glutamatergic transmission. To characterize the anatomical organization of excitatory signaling elements within this network, we analyzed the distribution, morphology, and colocalization of the AMPA receptor subunit GluA2 and the NMDA receptor subunit NR1, together with the postsynaptic scaffold protein PSD95. We also used immunofluorescence and confocal microscopy to examine the spatial relationship between GluA2-positive puncta and the astrocytic glutamate transporter EAAT2 in the preBötC of adult male rats. NR1-positive puncta were more abundant and densely distributed than GluA2-positive puncta, whereas GluA2–NR1 colocalized puncta constituted only a small fraction of either receptor population. NR1-positive puncta also showed limited colocalization with PSD95-positive puncta, indicating that a substantial fraction of NR1 immunoreactivity is not associated with PSD95-defined postsynaptic domains under the present imaging conditions. In contrast, GluA2-positive puncta displayed a non-random spatial proximity with EAAT2 astrocytic profiles and were frequently located within submicron distances. Together, these findings reveal a spatially differentiated organization of glutamatergic elements in the preBötC, characterized by abundant non-PSD95-associated NR1-positive puncta and close apposition between GluA2-positive puncta and EAAT2-immunoreactive astrocytic profiles. This organization provides an anatomical framework for future studies testing how glutamate receptor localization and glutamate clearance mechanisms contribute to excitability and respiratory rhythm generation and modulation.
The Hippo signaling pathway regulates the development of multiple organs, yet its role in the maturation of adult Leydig cells and testosterone synthesis is unclear. In this study, by integrating bulk RNA sequencing and immunohistochemistry, we characterized the spatiotemporal expression patterns of YAP and TEAD family members during postnatal Leydig cell development. Functional assays combining pharmacological inhibition, genetic knockdown, and CUT Tag-seq analysis were employed to elucidate the underlying mechanisms, and single-cell transcriptomic data from patients with idiopathic non-obstructive azoospermia (iNOA) were analyzed to assess clinical relevance. Results of single-cell transcriptomic analysis revealed that YAP, TEAD1, and TEAD3 are significantly elevated in Leydig cells from patients with iNOA compared with normal controls. In contrast, under normal physiological conditions, YAP protein levels progressively declined during Leydig cell maturation into adulthood, accompanied by increased phosphorylation (p-YAP). Correspondingly, Tead1 and Tead2 are markedly downregulated as immature Leydig cells differentiate into adult Leydig cells. Functional assays further revealed that knockdown of either YAP or TEAD and K975 treatment significantly increased testosterone synthesis and upregulated the expression of Hsd3b6, Cyp17a1, and Hsd17b3. Mechanistically, the YAP/TEAD complex may transcriptionally repress these steroidogenic genes by restricting the deposition of the active histone mark H3K27ac at their promoter regions. Our findings demonstrate that YAP/TEAD orchestrates postnatal Leydig cell maturation by epigenetically repressing the steroidogenic program, revealing a novel role for the Hippo pathway in male reproductive development and providing insights into the pathogenesis of iNOA.
Obesity and endocrine disruptors (EDs) promote endoplasmic reticulum (ER) stress, a mechanism associated with cardiovascular diseases. Among EDs, BPS is widely used in consumer products. Whether BPS promotes cardiac ER stress, either alone or combined with a high-fat diet, remains unclear. Male C57BL/6 mice were assigned to a standard chow diet (SC—15 kJ/g), SC+BPS (SCB), high-fat diet (HF—21 kJ/g), or HF+BPS (HFB), receiving BPS (25 μg/kg/day) through their drinking water for 12 weeks. Body mass (BM), lipid profile, cardiac risk ratio, heart mass, and cardiac ER stress, apoptosis, and oxidative stress markers were assessed. A high-fat diet intake increased BM gain, total cholesterol, cardiac risk ratio, and heart mass compared with standard chow diet intake. These parameters were higher in the SCB group than in the SC group. ER stress (GRP78, ATF4, and CHOP protein expression), pro-apoptotic (BAX protein expression and caspase 3 immunostaining), and pro-oxidative (NOX2 and NOX4 protein expression as well as MDA levels) markers were elevated in high-fat diet-fed animals compared with standard chow-fed animals. BPS exposure increased these parameters in standard chow-fed animals. High-fat diet intake reduced BCL2 expression and increased the BAX/BCL2 ratio compared with standard chow diet intake. BPS exposure increased BCL2 expression in standard chow-fed animals and increased the BAX/BCL2 ratio in high-fat diet-fed animals. ER stress appears to play a role in the cardiac effects of BPS. BPS exposure exacerbated the high-fat diet-induced pro-apoptotic response.
The staining performance of hemalum solutions is critically dependent on pH, yet the acetic acid content varies among different hemalum formulations. This study aimed to clarify the relationship between the acetic acid ratio (pH) of Gill’s hemalum and optimal nuclear staining, and to improve the hematoxylin and eosin (H E) staining method. Gill’s hemalum with varying acetic acid concentrations was used for H E staining of surgical and biopsy specimens. Optimal nuclear staining was achieved at 3.48
The measurement of intracellular reactive oxygen species (ROS) levels provides critical insights into cell health and pathophysiology. Here, we present a semi-quantitative, plate reader-based protocol using CellROX Green that can be used for the real-time measurement of general ROS levels of viable suspension mammalian cells. With menadione being the oxidative stress inducer, a cell concentration of 100,000 cells per well, a CellROX Green concentration of 10 µM, and a CellROX Green incubation time of 1 h was shown to provide the most accurate measurement of intracellular ROS levels in viable human monocyte leukemia (THP-1) cells. Unlike plate reader protocols using other general ROS-sensitive fluorogenic probes, this plate reader protocol eliminates the washing step of the unbound probe prior to measuring fluorescence, making it more suitable for suspension cells. Using the presented protocol, the fluorescence of up to 96 samples can be measured in under 1 min, making it ideal for rapid screening applications where moderate to large differences in oxidative stress levels are to be measured.
Indirect flight muscles (IFMs) of Drosophila melanogaster are among the most structurally ordered and metabolically demanding tissues in the animal kingdom. Importantly, IFMs comprise two anatomically and mechanistically distinct muscle groups: the dorsal longitudinal muscles (DLMs) and the dorsoventral muscles (DVMs). Their ability to sustain high-frequency, stretch-activated oscillations depends not only on specialised sarcomeric architecture but on multilayered signalling networks that coordinate lineage specification, myoblast proliferation, fibre patterning, mechanotransduction, metabolic adaptation and adult maintenance. Although structural components of IFMs have been extensively characterized, less attention has been given to the signalling axis that integrates developmental cues with post-developmental function. Here, we synthesise current knowledge of the signalling pathways that orchestrate muscle development from embryogenesis through metamorphosis and into adult life. We discuss how canonical pathways, including Notch, Wingless (Wg), Hedgehog (Hh), fibroblast growth factor/Heartless (FGF/Htl), epidermal growth factor (EGF) Ras/MAPK, Rho-family GTPases, integrin complexes, Hippo and Transforming Growth Factor-beta (TGF-β), are repurposed and cross-regulated in the context of the DLMs and DVMs. We further examine how muscle identity genes interface with signalling inputs to define fibre-type specification. Finally, we highlight that mechanical, metabolic and growth-regulatory pathways form an integrated signalling hub that tunes muscle size, power output and endurance. This pathway-centric perspective reframes IFM development as a systems-level template of signal integration and provides a foundation for further understanding of tissue morphogenesis.
Chromatin contains genetic information in eukaryotes. Multiple epigenetic mechanisms, such as chemical modifications of DNA and histone post-translational modifications (PTMs), regulate chromatin organization and architecture, influencing RNA transcription and driving diverse gene expression patterns from the same genome. This epigenetic control is highly dynamic and influenced by various events, such as DNA insults which cause altered gene expressions to drive aging, neurodegeneration, and cancer. However, this interplay remains poorly understood and identifying novel molecular effectors may shed light for therapeutic purposes. Here, we investigate the consequences of UV exposure in cells expressing a mutant form of damage-specific DNA-binding protein 2 (DDB2), a key protein for UV damage repair, in which interaction with PCNA is disrupted, thereby impairing proper DDB2 degradation. Our data reports that overexpression of mutant DDB2 resulted in persistent epigenetic alterations after UV irradiation, which led to elevated detection of H3K9 acetylation and trimethylation, as well as increments in 5-methylcytosine and altered subnuclear localization of γ-H2AX. Collectively, these findings demonstrate that DDB2–PCNA interaction regulates not only DDB2 stability, but also the epigenetic landscape of the genome.
The chloride (Cl-) channel ClC-5 is a vesicular Cl-/H+ exchanger belonging to the CLC family of voltage-gated Cl- channels. In mammals and humans, ClC-5 is involved in protein endocytosis and intracellular vesicular transport in the proximal tubule (PT) of the kidney. ClC-5 has also been identified in the amphibian kidneys, but little is known about its intrarenal distribution and physiological role. Our study aimed to perform a morphofunctional characterization of ClC-5 in the kidneys of the grass frog (Rana temporaria) and the lake frog (Pelophylax ridibundus) during receptor-mediated endocytosis, as well as under conditions of hypernatremia, dehydration, and hypervolemia. We used methods of immunocytochemistry, immunohistochemistry, confocal microscopy, and quantification of fluorescent signal intensity. The distribution of ClC-5 along the nephron and its intracellular pattern in glomerular and tubular cells were demonstrated. The colocalization of ClC-5 and the endocytic receptor megalin in the same PT cell compartments, together with a time-dependent increase in ClC-5 amount during lysozyme reabsorption, indicates the involvement of this channel in receptor-mediated endocytosis in frogs. ClC-5 expression in epithelial cells was increased in dehydrated and hypernatremic frogs, but not in hypervolemic ones. Hypernatremia and hypervolemia led to an increased number of ClC-5-containing glomeruli. These results demonstrate that the renal expression pattern of ClC-5 is similar in frogs and mammals. Increased ClC-5 expression is associated with functional changes resulting from modifications of epithelial transport owing to renal osmotic and ionic regulation.
Children conceived through assisted reproductive technologies (ART) are more sensitive to asthma and allergic respiratory diseases compared with children born through natural pregnancy. Growing evidence suggests that Toll-like receptors (TLRs) play a role in asthma. We previously demonstrated that TLR9 expression is significantly downregulated in the lung tissue of mouse fetuses generated through ART. The present study therefore investigated whether altered expression of TLR9 is maintained in the lung tissue in the perinatal period and in adult mice conceived through in vitro embryo culture and embryo transfer. We also examined the expression pattern of TLR9 in lung tissue. The study comprised one control (CG) and one experimental group (EG). Mice comprising EG were generated through the transfer of in vitro-derived F2 blastocysts. Mice obtained from naturally ovulated females served as the CG. Quantitative real-time PCR (qRT-PCR) and immunohistochemistry/immunofluorescence analyses were employed to determine the expression of TLR9. qRT-PCR analyses demonstrated that expression of TLR9 decreased 1.38-fold in EG when compared with the CG. Immunohistochemistry/immunofluorescence analyses revealed that the expression pattern of TLR9 is similar between the groups. Simultaneous staining of sequential sections further elucidated that TLR9 is expressed in type II alveolar epithelial cells. It therefore appears that altered expression of TLR9 observed in the lung tissue of mouse fetuses generated through embryo culture and embryo transfer persists into adulthood. To what extent the findings of the present study apply to human ART warrants further investigations.
Increased matrix stiffness is a key physical signal affecting the migration of hepatocellular carcinoma (HCC) cells, and mitochondrial dynamics and function also play important roles in cell migration. Plectin may influence mitochondrial dynamics and function through its cytoskeletal cross-linking function. However, the relationship between these two factors remains unclear. HCC cells were seeded on hydrogels with stiffness of 7 kPa and 53 kPa, respectively, to investigate the effects of matrix stiffness on plectin expression, mitochondrial dynamics and function, and cell migration. Moreover, plectin was knocked down to further assess its specific impacts on mitochondrial dynamics and function, as well as cell migration under different matrix stiffness. Compared with 7 kPa, high matrix stiffness (53 kPa) promotes HCC cell migration by upregulating plectin expression, promoting mitochondrial fusion, and enhancing mitochondrial function. Under high matrix stiffness, plectin knockdown weakens mitochondrial fusion capacity and function, reducing cell migration. Subsequently, we treated cells with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to inhibit mitochondrial function. This treatment significantly suppressed cell migration on high- matrix stiffness. Then, when mitochondrial dynamics were disrupted by the mitochondrial fusion inhibitor 8 (MFI8), mitochondrial function was compromised, and cell migration decreased. High matrix stiffness enhances mitochondrial function by driving mitochondrial fusion through increasing plectin expression, thereby promoting the migration of HCC cells. It provides new insights into the mechanobiological mechanisms underlying matrix stiffness affected HCC cell migration.
Oxidative stress and inflammation, in particular, are crucial factors in the pathogenesis of myocardial infarction (MI). Isoproterenol (ISO) is frequently preferred for creating experimental MI models because it reflects the pathophysiology of MI in humans. The purpose of this study was to identify potential alterations in OTULIN and progranulin (PRGN) levels at various stages during and following cardiac remodeling in experimental MI caused by ISO. The study consisted of control groups and ISO groups (ISO-6-h, ISO-24-h, ISO-3-day, and ISO-7-day) representing important time points in experimental MI and subsequent cardiac remodeling. The experimental MI model induced by ISO was validated by increased serum cardiac markers and histopathological changes in the heart tissue. Furthermore, it was determined that oxidative stress and inflammation, important factors in MI pathophysiology, emerged in the heart tissue after ISO administration. However, in the ISO-7-day group, a significant reduction in oxidative stress and inflammation was observed along with cardiac remodeling. In the experimental MI model, a time-dependent decrease in cardiac OTULIN levels was observed, while conversely, an increase in cardiac PRGN levels was detected. These time-dependent dynamic changes in cardiac OTULIN and PRGN suggest that both may play a role in processes related to MI and cardiac remodeling. In conclusion, endogenous OTULIN and PRGN may be associated with oxidative stress and inflammation in cardiac remodeling during and after experimental MI. This reveals the potential of OTULIN and PRGN as cardiac biomarkers in MI prognosis and suggests they could also be molecular targets for therapeutic strategies.
Eosinophils are increasingly recognized as contributors to tissue remodeling and chronic inflammation, yet their spatiotemporal behavior in sterile granulomatous reactions is incompletely defined. We performed a day 1–14 histopathological time course of a reproducible sterile granuloma induced by implantation of a heat-coagulated egg white pellet into the murine peritoneum. The reaction showed a biphasic inflammatory pattern with early neutrophil predominance followed by a sustained eosinophil-rich phase that persisted through late remodeling. Across time points, lesions progressed from exudative inflammation with prominent fibrin to organized fibroplasia and neovascularization, culminating in collagen-dominated encapsulation by day 14. Eosinophils concentrated at the pellet–tissue interface, frequently displaying apoptosis and degranulation, and occurring in close spatial association with fibroblasts and macrophages. At late time points, the outer granuloma zone contained mononuclear phagocyte/macrophage-like cells alongside dense connective tissue maturation. Together, these findings provide a temporal histopathology atlas of sterile implant granuloma maturation and introduce a semiquantitative framework that summarizes key tissue-composition transitions (fibrinous exudate → fibroplasia/collagen encapsulation) alongside eosinophil enrichment at the pellet–tissue interface.
Lipofuscin is a subcellular pigmented granule that has long been considered a sign of wear-and-tear and aging. This granule cannot be degraded but may be diluted by cell division. In the heart, lipofuscin can be easily identified in cardiomyocytes where proliferative capacity is limited, and to a lesser extent in other cardiac cell types, by their characteristic morphological features. They appear as yellow–brown granules in histopathology sections reflecting their lipid origins, and as heterogenous membrane-bound bodies containing a mix of lipid and electron-lucent content in transmission electron microscopy. Lipofuscin was originally considered a mere byproduct of age-related cell senescence but studies over the past three decades have indicated that its role and mechanism of formation may be far more complex. In this review, we examine how lipofuscin is formed and its implications in aging and diseases of the heart. While the focus of this review is on lipofuscin in the two most predominate cell types of the heart, cardiomyocytes and fibroblasts, we also touch on the presence of lipofuscin in the vasculature in certain disease phenotypes that are directly relevant to cardiovascular health.
In this review, we summarize the data on the cellular pigment lipofuscin that accumulates in liver tissue over time, due to aging and cellular stress. Despite the presence of these typical subcellular inclusions under various conditions, relatively little is known about their origins, roles, and effects on liver cell and tissue health. Pathologists use the presence of lipofuscin, in combination with other markers, to achieve differential diagnosis across various diseases. Routine histological stains reveal characteristic irregular shaped intracellular inclusions of lipofuscin that cannot be missed. Moreover, lipofuscin is autofluorescent and in transmission electron microscopy it appears in the cytoplasm as irregularly shaped structures containing fat and floccular material with varying electron density. Herein, we discuss the current state of knowledge concerning the origin and function of this pigment in the liver. Lipofuscin can distinctively be found in liver, although it has also been reported in cells in the heart, brain, and eye. Its biochemical composition is heterogeneous and varies depending on the tissue and the age of the organism. The liver parenchymal cells have efficient cellular waste disposal mechanisms, but they are still susceptible to aging. Lipofuscin accumulation in the liver may result from ongoing oxidative damage and impaired hepatic detoxification leading to cellular stress.
Cornification is a terminal differentiation process involving the synthesis of structural proteins, including α-keratin, loricrin and filaggrin-like protein, with the enzymatic contribution of transglutaminase 1 (TGM-1). The present study aimed to characterize the expression profiles of these proteins in the ortho- and parakeratinized epithelia of the lingual mucosa in domestic goose embryos from day 9 to day 25 of incubation, using immunohistochemical analysis. The spatial expression of the structural proteins and TGM-1 in both lingual epithelia was analyzed during three developmental stages: embryonic, transformation, and prehatching, revealing a slight temporal shift. During the embryonic stage, the expression of structural proteins and TGM-1 began, indicating early epithelial cytodifferentiation. At the transformation stage, differentiation of the epithelia into three layers and periderm formation with a marked increase in expression of the studied proteins occurred. For the first time, differences in the expression patterns of structural proteins and TGM-1 were observed in the periderm. By the prehatching stage, a cornified layer was present in both epithelia. Notably, only α-keratin expression in both epithelia and filaggrin-like protein and loricrin expression in cornified layer of the orthokeratinized epithelium resembled the adult pattern. These findings indicate that cornification is initiated during the transformation stage, approximately two-thirds of the embryonic period. Before hatching, epithelia have a cornified layer. However, the expression patterns of structural proteins and TGM-1 differ from those observed in adults, suggesting that cornification continues after hatching.
