Effects of Tcte1 knockout on energy chain transportation and spermatogenesis: implications for male infertility.

STUDY QUESTION: Is the Tcte1 mutation causative for male infertility? SUMMARY ANSWER: Our collected data underline the complex and devastating effect of the single-gene mutation on the testicular molecular network, leading to male reproductive failure. WHAT IS KNOWN ALREADY: Recent data have revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males, including dysplasia of fibrous sheath (DFS) and multiple morphological abnormalities in the sperm flagella (MMAF). The nexin-dynein regulatory complex (N-DRC) coordinates the dynein arm activity and is built from the DRC1-DRC7 proteins. DRC5 (TCTE1), one of the N-DRC elements, has already been reported as a candidate for abnormal sperm flagella beating; however, only in a restricted manner with no clear explanation of respective observations. STUDY DESIGN SIZE DURATION: Using the CRISPR/Cas9 genome editing technique, a mouse Tcte1 gene knockout line was created on the basis of the C57Bl/6J strain. The mouse reproductive potential, semen characteristics, testicular gene expression levels, sperm ATP, and testis apoptosis level measurements were then assessed, followed by visualization of N-DRC proteins in sperm, and protein modeling in silico. Also, a pilot genomic sequencing study of samples from human infertile males (n = 248) was applied for screening of TCTE1 variants. PARTICIPANTS/MATERIALS SETTING METHODS: To check the reproductive potential of KO mice, adult animals were crossed for delivery of three litters per caged pair, but for no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild-type (WT, control group), heterozygous Tcte1+/- and homozygous Tcte1-/- male mice. Gross anatomy was performed on testis and epididymis samples, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) was done for mice testis tissues. STRING interactions were checked for protein-protein interactions, based on changed expression levels of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence in situ staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the amount of ATP in spermatozoa, the luminescence level was measured. In addition, immunofluorescence in situ staining was performed to check the level of apoptosis via caspase 3 visualization on mouse testis samples. DNA from whole blood samples of infertile males (n = 137 with non-obstructive azoospermia or cryptozoospermia, n = 111 samples with a spectrum of oligoasthenoteratozoospermia, including n = 47 with asthenozoospermia) was extracted to perform genomic sequencing (WGS, WES, or Sanger). Protein prediction modeling of human-identified variants and the exon 3 structure deleted in the mouse knockout was also performed. MAIN RESULTS AND THE ROLE OF CHANCE: No progeny at all was found for the homozygous males which were revealed to have oligoasthenoteratozoospermia, while heterozygous animals were fertile but manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of Tcte1 mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing or linked with apoptosis or spermatogenesis. In Tcte1-/- males, the protein was revealed in only residual amounts in the sperm head nucleus and was not transported to the sperm flagella, as were other N-DRC components. Decreased ATP levels (2.4-fold lower) were found in the spermatozoa of homozygous mice, together with disturbed tail:midpiece ratios, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at a similar level in all three mouse genotypes. Mutation screening of human infertile males revealed one novel and five ultra-rare heterogeneous variants (predicted as disease-causing) in 6.05% of the patients studied. Protein prediction modeling of identified variants revealed changes in the protein surface charge potential, leading to disruption in helix flexibility or its dynamics, thus suggesting disrupted interactions of TCTE1 with its binding partners located within the axoneme. LARGE SCALE DATA: All data generated or analyzed during this study are included in this published article and its supplementary information files. RNAseq data are available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE207805. The results described in the publication are based on whole-genome or exome sequencing data which includes sensitive information in the form of patient-specific germline variants. Information regarding such variants must not be shared publicly following European Union legislation, therefore access to raw data that support the findings of this study are available from the corresponding author upon reasonable request. LIMITATIONS REASONS FOR CAUTION: In the study, the in vitro fertilization performance of sperm from homozygous male mice was not checked. WIDER IMPLICATIONS OF THE FINDINGS: This study contains novel and comprehensive data concerning the role of TCTE1 in male infertility. The TCTE1 gene is the next one that should be added to the 'male infertility list' because of its crucial role in spermatogenesis and proper sperm functioning. STUDY FUNDING/COMPETING INTERESTS: This work was supported by National Science Centre in Poland, grants no.: 2015/17/B/NZ2/01157 and 2020/37/B/NZ5/00549 (to M.K.), 2017/26/D/NZ5/00789 (to A.M.), and HD096723, GM127569-03, NIH SAP #4100085736 PA DoH (to A.N.Y.). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Pattern Distribution of Connexins in the Ortho- and Parakeratinized Epithelium of the Lingual Mucosa in Birds.

Connexins are important proteins involved in cell-to-cell communication and cytodifferentiation during renewal and cornification of the multilayered epithelia. So far, there is a lack of reports on this subject in birds' structurally different ortho- and parakeratinized epithelium of the tongue. The study aims to describe the distribution and expression profiles of the α-connexins (Cx40 and 43) and ß-connexins (Cx26, 30, and 31) in those epithelia in duck, goose, and domestic turkey. Research revealed the presence of the mentioned connexins and the occurrence of interspecies differences. Connexins form gap junctions in the cell membrane or are in the cytoplasm of keratinocytes. Differences in connexin expression were noted between the basal and intermediate layers, which may determine the proliferation of keratinocytes. Cx40, 43, and Cx30 in the gap junction of the keratinocytes of the intermediate layer are related to the synchronization of the cornification process. Because of the exfoliation of cornified plaques, a lack of connexins was observed in the cornified layer of orthokeratinized epithelium. However, in parakeratinized epithelium, connexins were present in the cell membrane of keratinocytes and thus maintained cellular integrity in gradually desquamating cells. The current studies will be useful in further comparative analyses of normal and pathological epithelia of the oral cavity in birds.

Morphogenesis of the uterine tube in the European shorthair cat in the prenatal and postnatal period: Light microscopy and three-dimensional reconstruction study.

The study aimed to describe the morphogenesis of the domestic cat uterine tube, from the primordial tubal segment of the paramesonephric duct (TsPD) to the mature organ. The specific aims included identifying differences in the rate and way of development of the layers in the wall of the infundibulum, the ampulla, and the isthmus. In the study we conducted light microscopic (LM) observations of the uterine tube wall, and analysis of 3D models, which were used for the first time to visualize the arrangement of the paramesonephric duct and highlight the pattern of the developing mucosal folds. The results revealed the two stages in the prenatal development of the domestic cat uterine tube. The first stage lasts from day 26-44 p.c. (post-conception) and includes the growth of the TsPD. It starts with formation of the TsPD's epithelial tubule in the mesenchyme of the anterior part of the mesonephros. The tubule, surrounded by mesenchyme, elongates posteriorly, and the TsPD can be divided into three sections according to their location and diameter, i.e., anterior, middle, and posterior. The first stage ends with the histodifferentiation of the mesenchyme into loose connective tissue and mucosa formation. The second stage lasts from approximately day 45-63 p.c. and comprises the formation of the proper layers in the uterine tube wall. The pace and pattern of layers' development are specific to each section of the uterine tube, as tissues first differentiate in its anterior part and then continue posteriorly. The mucosal folds appear after day 44 p.c. in the infundibulum, and they become long and branched by the end of the prenatal period. Then, approximately day 48 p.c., the single mucosal folds appear in the ampulla, and at the end, i.e., about day 63 p.c., they become visible in the isthmus. The 3D models revealed the unique spiral-shaped arrangement of the mucosal folds in the ampulla. The tunica muscularis starts to develop ca day 48-51 p.c. in the anterior part of the uterine tube. On approximately day 55 p.c., the circular muscle layer can be distinguished in the ampulla, while in the isthmus, it develops just before birth. The postnatal development of the infundibulum includes the formation of the labyrinth-like system of mucosal folds, whereas in the ampulla and isthmus, branched mucosal folds and the second longitudinal muscle layer develop postnatally. At the end of the prenatal period, the differences in the wall thickness between the individual segments of the uterine tube reflect the proper proportions of the uterine tube wall in the mature female.

Alpha-Keratin, Keratin-Associated Proteins and Transglutaminase 1 Are Present in the Ortho- and Parakeratinized Epithelium of the Avian Tongue.

The lingual mucosa in birds is covered with two specific types of multilayered epithelia, i.e., the para- and orthokeratinized epithelium, that differ structurally and functionally. Comprehensive information on proteins synthesized in keratinocyte during their cytodifferentiation in subsequent layers of multilayered epithelia in birds concerns only the epidermis and are missing the epithelia of the lingual mucosa. The aim of the present study was to perform an immunohistochemical (IHC) and molecular analysis (WB) of bird-specific alpha-keratin, keratin-associated proteins (KAPs), namely filaggrin and loricrin, as well as transglutaminase 1 in the para- and orthokeratinized epithelium covering the tongue in the domestic duck, goose, and turkey. The results reveal the presence of alpha-keratin and KAPs in both epithelia, which is a sign of the cornification process. In contrast to the epidermis, the main KAPs involved in the cornification process of the lingual epithelia in birds is loricrin. Stronger expression with KAPs and transglutaminase 1 in the orthokeratinized epithelium than in the parakeratinized epithelium may determine the formation of a more efficient protective mechanical barrier. The presence of alpha-keratin, KAPs, and transglutaminase 1 epitopes characteristic of epidermal cornification in both types of the lingual epithelia may prove that they are of ectodermal origin.

Three-dimensional characteristic of fungiform papillae and its taste buds in European bison (Bison bonasus), cattle (Bos taurus), and Bison bonasus hybrid.

BACKGROUND: Our recent macro- and scanning electron microscopic study of tongue conducted on domesticated cattle, wild living European bison, and Bison bonasus hybrid revealed various spatial arrangement and number of gustatory and mechanical papillae between parental species and their hybrid. Furthermore, scanning electron microscopy analysis of gustatory papillae indicated the variable distribution of fungiform papillae (Fu) over the surface of the tongue, which could be significant in differentiated taste perception during feeding in studied wild living and domesticated husbandry ruminants. To specify the detailed microstructure of Fu papillae with connective tissue cores (CTC) and intraepithelial taste buds system, the first time the three-dimensional computer-aided analysis of serial histoslides resulted in the rendering of 3D reconstructions of Fu papillae. RESULTS: The comparative analysis of 3D models Fu papillae conducted in six areas of lingual mucosa of each tongue revealed information about, microstructural diversity of Fu papillae in studied ruminants. The estimation of number and density of Fu papillae on tongues, rate of protrusion of papillae over mucosa, and a number of taste buds per papilla allowed to state the ventral surface of the lingual apex and posterolateral surfaces of the lingual torus as regions important in taste perception, as in the preselection of taken food, as well in the analysis of food during rumination, respectively. On the 3D models were observed three structural types of CTC of different distribution on the tongue in studied species. The quantitative data of the number of taste buds on Fu papillae have regional functional differences in the taste system important in feeding and veterinary practice. Moreover, our analysis determined specific features in examined hybrid and showed similarities of some studied features with cattle, i.e., maternal species. CONCLUSIONS: The 3D reconstruction method used for the first time in the field of study of the lingual papillae and taste buds system can be considered as an innovative and effective tool in assessing of the microstructures of Fu papillae, and it could be suitable for further studies of taste system structures in normal and pathological condition.

Unique pattern of histogenesis of the parakeratinized epithelium on lingual prominence in the domestic goose embryos (Anser anser f. domestica).

A triangular lingual prominence (LP) is a characteristic part of the tongue in Anseriformes containing adipose tissue. The parakeratinized epithelium (PEp) covers the LP. Studies aimed to describe the histogenesis of PEp during the process of the intensive formation of the LP in domestic goose during embryonic period and to determine the structural readiness to perform a protective function. The study were conducted by using LM, SEM and TEM technique. The results revealed that on day 16th the undifferentiated epithelium of LP transformed into the typical avian multilayered epithelium. Contrary to pattern of histogenesis of parakeratinized epithelium on the lingual body, on the medial and lateral areas of the elongating and bulging LP were formed epithelial furrows. Which around 20th day, on lateral areas of LP deepened up to half of epithelium, whereas on the medial area began to fade. The ultrastructure of cells lying in furrows indicated progressive apoptosis-like degeneration. On the 25th day, shallow furrows were only present on lateral areas, where bulging of LP was continued. Whereas the epithelium on medial area started cornification by the accumulation of cytokeratin fibers. Lack of the periderm during the development of the PEp of the LP indicated its endodermal origin.

Evaluating Sexual Dimorphism of the Muscle Spindles and Intrafusal Muscle Fibers in the Medial Gastrocnemius of Male and Female Rats.

This study sought to investigate the sexual dimorphism of muscle spindles in rat medial gastrocnemius muscle. The muscles were cut transversely into 5-10 and 20 µm thick serial sections and the number, density, and morphometric properties of the muscle spindles were determined. There was no significant difference (p > 0.05) in the number of muscle spindles of male (14.45 ± 2.77) and female (15.00 ± 3.13) rats. Muscle mass was 38.89% higher in males (1.08 vs. 0.66 g in females), making the density of these receptors significantly higher (p < 0.01) in females (approximately one spindle per 51.14 mg muscle mass vs. one per 79.91 mg in males). There were no significant differences between the morphometric properties of intrafusal muscle fibers or muscle spindles in male and female rats (p > 0.05): 5.16 ± 2.43 and 5.37 ± 2.27 µm for male and female intrafusal muscle fiber diameter, respectively; 5.57 ± 2.20 and 5.60 ± 2.16 µm for male and female intrafusal muscle fiber number, respectively; 25.85 ± 10.04 and 25.30 ± 9.96 µm for male and female shorter muscle spindle diameter, respectively; and 48.99 ± 20.73 and 43.97 ± 16.96 µm for male and female longer muscle spindle diameter, respectively. These findings suggest that sexual dimorphism in the muscle spindles of rat medial gastrocnemius is limited to density, which contrasts previous findings reporting differences in extrafusal fibers diameter.

Microstructural changes of the wall of uterine horn in the domestic cat (Felis silvestris catus) during postnatal development.

The study describes the details of histological changes occurring during the postnatal development of the wall of the uterine horn in the European shorthair domestic cat. Light microscope observations and morphometric analysis were carried out to investigate the rate of development of the wall of the uterine horns in order to estimate the maturing time. Uterine horns of newborn and 1week-old cats are fetal-like and showing smooth, nonfolded endometrium lined with pseudostratified epithelium, deprived of uterine glands, and poorly developed two-layered myometrium. The lamina propria of the mucosa becomes folded in the first month of postnatal life. At the same time, the primordia of the uterine glands appear, elongating, doubling in number and reaching the basal layer by sixth month of age. In six-month-old cats, the lamina propria of the mucosa is formed into the functional and basal layer and its thickness increases rapidly. Until the sixth month of age, the inner circular muscle layer is the widest part of the myometrium, the outer longitudinal layer is the thinnest, while the width of the vascular layer is an intermediate value between the width of the inner and outer layer. From the seventh month of age, the proportion of the thickness of the inner and outer layer is reversed and the vascular layer becomes the thinnest part of the myometrium. Between seventh and twelfth month of age, the structure of the wall of the uterine horn does not change significantly, as the longitudinal mucosal folds limiting the narrow lumen of the uterine horns, and the arrangement of the layers in the wall is assigned to a pattern according to which the endometrium represents about 40% of the total width of the wall, while the myometrium is about 60%. In the studied time span of postnatal life, the average thickness of the wall of the uterine horn increases 9.4 -fold, the thickness of the lamina propria and mucosal folds increases 6.9-fold and 3.8-fold, respectively, while the thickness of the myometrium increases 17.3-fold. Summing up, the process of maturation of the wall of the uterine horns in the domestic cat lasts up to sixth month of postnatal life and includes the formation of the endometrial longitudinal folds, appearance and development of the uterine glands, division of the lamina propria of the mucosa into the functional and basal layers, and continued development of the circular, vascular and longitudinal muscle layers.

Functional morphology of the tongue in the domestic turkey (Meleagris gallopavo gallopavo var. domesticus).

Nowadays, microstructural and ultrastructural analysis of organs of the avian beak cavity points to new aspects of adaptation to food intake through the various feeding groups. These data should undoubtedly be considered in the time of mass production of compound feed in poultry, when many studies analyze the optimal size of food particles and their doses. Galliformes possess complex mechanisms of food collection and transport in the beak cavity. They collect food by pecking and transport food by using catch-and-throw and slide-and-glue mechanisms. The aim of current research is to conduct functional analysis of the tongue in poultry such as domestic turkey in context of type of food, method of food intake, and transport to the esophagus. The study involves observations of macroscopic and microscopic structures of the tongue mucosa by light microscopy and scanning electron microscopy techniques with histochemical analysis of lingual glands. The obtained results showed that the tongue in domestic turkey fills two-thirds of the beak cavity. The lingual structure responsible for pecking is a rigid plate called lingual nail that works similar to a shovel to collect food. The median groove presented on surface of the tongue indicated path of food transport. The conical papillae on border between the lingual body and root are responsible for the last stage of food transport, while the papillae on the sides of root stabilize the path of food transport. For the first time, the presence of 2 types of cornified mucosal epithelia, orthokeratinized and parakeratinized epithelium, was presented. The analysis of occurrence of complex tubular lingual glands indicates production of mucous secretions composed of neutral mucopolysaccharides, with addition of sialomucins and sulfomucins. Mucous secretions moisturize surface of the tongue, thus facilitating the transport of dry food. The presence of sulfur mucopolysaccharides responds to protective function. To sum up, the tongue in domestic turkey is adapted to collect fine or coarse ground feed in form of mash or pellets through pecking and its transport to the esophagus using the slide-and-glue and throw-and-catch mechanisms.

Macro- and microscopic study on the tongue and lingual papillae of Bison bonasus hybrid as an interspecific species (Bos taurus × Bison bonasus).

Nowadays the processes of crossing of different mammalian species are well characterized in light of genetics, and possibility of reproduction. Generally, lack detailed studies on changes in anatomy and histology of internal systems of hybrids. Bison bonasus hybrid is an interspecific species between the bull of European bison (Bison bonasus), and female of domesticated cattle (Bos taurus). To study the anatomy of tongue and distribution of lingual papillae on Bison bonasus hybrid tongue, a stereomicroscopic and scanning electron microscopic observations was conducted. We aimed to study the appearance of macro- and microscopic traits in relation to parental species and other bovids. Results indicate that Bison bonasus hybrid tongue possesses characteristic traits for parental species and ruminants from Bovidae family and also species-specific features differ from mentioned animals. Specific new traits in hybrid are: V-shaped arrangement of lingual papillae on ventral surface of the tongue, rosette arrangement of conical papillae on the top of lingual prominence, bigger number of vallate papillae and closer arrangement of vallate papillae. In hybrid observed also the appearance of combination of parental traits. Similar to European bison are rectangular shape of lingual prominence and absence of filiform papillae on the root of tongue. Presence of lateral processes of filiform papillae on the apex of tongue, and arrangement of vallate papillae into two rows corresponded to cattle. Development of new morphological features of tongue and its mucosal papillae in Bison bonasus hybrid opens new issue for future studies on organs of digestive system in animals after hybridization.

Innervation of internal female genital organs in the pig during prenatal development.

This study investigated the innervation of internal genital organs in 5-, 7- and 10-week-old female pig foetuses using single and double-labelling immunofluorescence methods. The structure and topography of the organs was examined using a scanning electron microscope (SEM). The investigations revealed differences in the innervation between the three developmental periods. Immunostaining for protein gene product 9.5 (PGP; general neural marker) disclosed solitary nerve fibres in the external part of the gonadal ridge and just outside of the mesenchyme surrounding mesonephric ducts in 5-week-old foetuses. Double-labelling immunohistochemistry revealed that nerve fibres associated with the ridge expressed dopamine ß-hydroxylase (DßH; adrenergic marker) or vesicular acetylcholine transporter (VAChT; cholinergic marker). In 7-week-old foetuses, the PGP-positive nerve terminals were absent from the gonad but some of them ran outside and along, and sometimes penetrated into the mesenchyme surrounding the tubal and uterine segments of the paramesonephric ducts and uterovaginal canal. Few axons penetrated into the mesenchyme. DßH-positive fibres were found in single nerve strands or bundles distributed at the edge of the mesenchyme. VAChT-positive nerve terminals formed delicate bundles located at the edge of the mesenchyme, and the single nerves penetrated into the mesenchyme. DßH was also expressed by neurons which formed cell clusters comprising also DßH- or VAChT-positive nerve fibres. In 10-week-old foetuses, PGP-positive nerve fibres were still absent from the ovary but some were distributed in the mesenchyme associated with the uterovaginal canal and uterine and a tubal segment of the paramesonephric ducts, respectively. DßH- or VAChT-positive nerve fibres were distributed at the periphery of the mesenchyme associated with the uterovaginal canal. Some DßH- and many VAChT-positive nerve fibres were evenly distributed throughout the mesenchyme. The clusters of nerve cells comprised DßH-positive perikarya and DßH- or VAChT-positive nerve fibres. The investigations revealed no DßH/VAChT-positive nerve fibres or neurons as well as no nerve structures stained for calcitonin gene-related peptide and/or substance P (sensory markers) associated with the genital organs in the studied prenatal periods.

Alpha-keratin and corneous beta protein in the parakeratinized epithelium of the tongue in the domestic goose (Anser anser f. domestica).

The parakeratinized epithelium is a common epithelium in the oral cavity in birds and is characterized by the presence of cell nuclei in the cells of the cornified layer. This epithelium covers almost the entire dorsal surface of the tongue in the domestic goose apart of the lingual nail and conical papillae. So far no study has identified the molecular proteins alpha-keratin (IF-keratin) and/or corneous beta protein (CBP), which are responsible for keratinization or cornification processes in the parakeratinized epithelium of domestic geese. The study was performed using immunohistochemical (IHC) methods to identify alpha-keratin. The innovative method of Raman microspectroscopy was used to determine the presence of CBP and specify their percentage in epithelial layers of the parakeratinized epithelium. The results revealed that alpha-keratin is present in the whole parakeratinized epithelium. A strong staining reaction was detected in the basal and intermediate layers and a less strong staining reaction in the cornified layer. Raman microspectroscopy analysis confirmed the presence of alpha-keratin and demonstrated that its percentage decreases from the basal layer to the cornified layer. The Raman microspectroscopy technique revealed the occurrence of CBP in the parakeratinized epithelium and demonstrated that the percentage of this protein increases from the basal layer to the cornified layer. Performed analysis determines that parakeratinized epithelium undergoes cornification. However, the lower percentage of CBP in the cornified layer of parakeratinized epithelium than in orthokeratinized epithelium points to the fact that parakeratinized epithelium has a weaker protective function.

Ultrastructural study of uterine epithelium in the domestic cat during prenatal development.

The study describes the sequence of ultrastructural changes of the endometrial lining epithelium, first in the primordial paramesonephric ducts, then in the differentiating uterine horns, and finally in the mature uterus. The research material comprised female fetuses of the domestic cat, aged 33-63 days post conceptionem (p.c.), and mature females. The ultrastructural observations of the developing epithelium were conducted using transmission electron microscopy (TEM) and light microscopy (LM). The results revealed two basic stages in the development of the uterine mucosal epithelium in the domestic cat: the development of the epithelium of the uterine segment of the paramesonephric ducts, lasting from day 33 to day 42 p.c., and the differentiation of this epithelium into the mucosal epithelium of the uterus, starting after day 42 p.c. and lasting until the end of the prenatal period. The epithelium of the uterine segments of the paramesonephric ducts is pseudostratified with a flat surface, all cells being characterized by the same distribution of desmosomes, ribosomes, rough endoplasmic reticulum (rER) cisternae, and mitochondria in the cytoplasm. The differentiating epithelium of the uterus after day 51 p.c. is pseudostratified with a varied height. In the epithelial cells there are increases of (i) the number and complexity of the junctional complexes, (ii) the number of invaginations of the lateral cell membranes, and (iii) the number of elongated mitochondria, and also there appear distended cisternae of the rER, lipid droplets and clusters of glycogen, which suggest increasing metabolism and secretory activity of the cells during the transformation of the epithelium.

Embryonic development of parakeratinized epithelium of the tongue in the domestic duck (Anas platyrhynchos f. domestica): LM, SEM, and TEM observations.

The parakeratinized epithelium is a common and widespread type of keratinized epithelium in the oral cavity in adult birds. In contrast to orthokeratinized epithelium, which mostly covers mechanical papillae and the lingual nail, parakeratinized epithelium covers almost the entire dorsal surface of the tongue in birds. The characteristic feature of parakeratinized epithelium is the presence of nuclei in the keratinized layer. The present study aimed to investigate for the first time the micro- and ultrastructural changes of parakeratinized epithelium during embryonic development and to assess the readiness of the epithelium to serve protective functions during food transport to the esophagus. Three developmental stages were distinguished: embryonic, transformation, and pre-hatching stages. The embryonic stage lasts from the 9th to the 14th day of incubation and the epithelium is composed of undifferentiated epithelial cells. The transformation stage lasts from the 15th to the 22nd day of incubation and the epithelium undergoes transformation into stratified epithelium consisting of basal, intermediate, and superficial layers. The characteristic feature of this stage is formation of the periderm with osmophilic granules. The pre-hatching stage starts on the 23rd day, and the epithelium with a fully developed keratinized layer resembles that of the epithelium in adult animals. No periderm was observed on the epithelial surface. It was confirmed that at the time of hatching the parakeratinized epithelium is fully differentiated and ready to fulfill its function during food transport. The presence of periderm is a common feature characteristic for para- and orthokeratinized epithelium in the oral cavity of birds. However, the formation of the keratinized/cornified layer is different for these two types of keratinized epithelia.

Morphology and topography of internal reproductive organs in the female cat during prenatal and postnatal development: Scanning electron microscope and three-dimensional reconstruction study.

The study describes the morphology and topography of internal reproductive organs in the domestic cat from the early prenatal period to maturity, using macroscopic and scanning electron microscope (SEM) observations with three-dimensional (3D) reconstructions. Fifty-seven female cat fetuses aged between the 27th and 63rd day postconception (p.c.), two newborn cats, three juveniles (3-month-old) cats, and three mature (12-month-old) cats were used in the study. The age of fetuses was determined on the basis of the growth curve for the domestic cat. The rudiments of cat ovaries develop on the ventral surface of the mesonephroi and within 30 days p.c. move to the sides of the abdominal cavity, which is similar to the position of the ovaries in the adult cat. The mesonephroi regress at about the 50th day p.c., when the residual mesonephric ducts are still found in the lower part of the body of the uterus. The paramesonephric ducts develop on the lateral surface of the mesonephroi and by the 45th day p.c., differentiate into the uterine tubes and the uterus. The arrangement of the paramesonephric ducts in the abdominal cavity changes from the U- to the V-shaped system. The final topography of the uterine tubes is established between the 54th and 60th day p.c., as the uterine tubes become convoluted. Before the 54th day p.c., the uterine horns undergo rapid elongation and convolution, forming the W-shaped system. By the third month of postnatal life, the uterine horns become straight, as in the adult cat.

Ultrastructural study on the embryonic development of the orthokeratinized epithelium and its cornified layer (lingual nail) on the ventral surface of the lingual apex in the domestic duck (Anas platyrhynchos f. domestica).

The lingual nail as the cornified layer of the orthokeratinized epithelium in birds is responsible for the collection of solid food by pecking. The aim of the present study is to determine the manner of orthokeratinized epithelium development and assess the degree of readiness of the epithelium to fulfill its mechanical function at hatching. Three developmental phases are distinguished, i.e. embryonic, transformation and pre-hatching stage. In the embryonic stage lasting until day 13 of incubation the epithelium is composed of several layers of undifferentiated cells. During the transformation stage, from day 14 to 20 of incubation, the epithelium becomes differentiated to form three layers. A characteristic feature is the formation of osmophilic granules in the superficial layer, referred to as periderm granules. Until the pre-hatching stage the fibrous cytoskeleton of epithelial cells and an impermeable epithelial barrier are gradually developed. In the pre-hatching stage, a cornified lingual nail is formed, while the periderm is exfoliated. At hatching the orthokeratinized epithelium and lingual nail are fully developed and ready to perform feeding activities. The presence of periderm, similarly as in the epidermis, indicates the ectodermal derivation of the oral cavity epithelium. Moreover, occurrence of osmophilic granules may be considered as evidence for the phylogenetic affinity of birds and reptiles.

The development of lingual glands in the domestic duck (Anas platyrhynchos f. domestica): 3D-reconstruction, LM, and SEM study.

The major salivary glands of birds develop by branching or elongation of the epithelial cords. The development of the minor salivary glands in form of the lingual glands has never been described. Among birds, only Anatidae have three types of the lingual glands: rostral, caudo-lateral, and caudo-medial lingual glands. The study aims to characterize the manner and rate of the lingual glands development in the domestic duck and their topographical arrangement relative to the hyoid apparatus. The study reveals that all three types of the lingual glands develop by branching. We describe five stages of the lingual glands development in the domestic ducks: prebud, initial bud, pseudoglandular, canalicular, and terminal bud stage. The pattern of the lingual glands development in birds is similar to that described for mammals, with the exception, that the terminal buds are formed at the same time as the lumen of the glands. Generally, the rostral lingual gland starts to branch earlier than the caudal lingual glands. The 3D-reconstruction shows the location and direction of lingual gland development relative to the entoglossal cartilage and basibranchial bone. Light microscopy and scanning electron microscopy allow to characterize the histogenesis of the embryonic epithelium into glandular epithelium. At a time of hatching only secretory units of caudal lingual glands resemble the secretory units of the adult domestic duck. The rostral and caudo-lateral lingual glands are arranged on the sides of the entoglossal cartilage and basibranchial bone and caudo-madial lingual glands are located over the basibranchial bone. We suggest that such an arrangement of the lingual glands in the domestic duck is important during food intake and responsible for reduction of friction and formation of food bites.

Localization of Alpha-Keratin and Beta-Keratin (Corneous Beta Protein) in the Epithelium on the Ventral Surface of the Lingual Apex and Its Lingual Nail in the Domestic Goose (Anser Anser f. domestica) by Using Immunohistochemistry and Raman Microspectroscopy Analysis.

The epithelium of the ventral surface of the apex of the tongue in most birds is specified by the presence of the special superficial layer called lingual nail. The aim of the present study is to determine the localization of the alpha-keratin and beta-keratin (corneous beta protein) in this special epithelium in the domestic goose by using immunohistochemistry staining and the Raman spectroscopy analysis. Due to lack of commercially available antibodies to detect beta-keratin (corneous beta protein), the Raman spectroscopy was used as a specific tool to detect and describe the secondary structure of proteins. The immunohistochemical (IHC) detections reveal the presence of alpha-keratin in all layers of the epithelium, but significant differences in the distribution of the alpha-keratin in the epithelial layers appear. The staining reaction is stronger from the basal layer to the upper zone of the intermediate layer. The unique result is weak staining for the alpha-keratin in the lingual nail. Applications of the Raman spectroscopy as a complementary method not only confirmed results of IHC staining for alpha-keratin, but showed that this technique could be used to demonstrate the presence of beta-keratin (corneous beta protein). Functionally, the localization of alpha-keratin in the epithelium of the ventral surface of the lingual apex provides a proper scaffold for epithelial cells and promotes structural integrity, whereas the presence of beta-keratin (corneous beta protein) in the lingual nail, described also as exoskeleton of the ventral surface of the apex, endures mechanical stress. Anat Rec, 300:1361-1368, 2017. © 2017 Wiley Periodicals, Inc.

Development of mechanical papillae of the tongue in the domestic goose (Anser anser f. domestica) during the embryonic period.

Three types of mechanical papillae, i.e., conical, filiform, and hair-like papillae, are present on the tongue in the domestic goose. Within conical papillae, we distinguish three categories: large and small conical papillae on the body and conical papillae on the lingual prominence. The arrangement of mechanical papillae on the tongue in Anseriformes is connected functionally with different feeding mechanisms such as grazing and filter-feeding. The present work aims to determine whether morphology of three types of mechanical papillae in goose at the time of hatching is the same as in an adult bird and if the tongue is prepared to fulfill feeding function. Our results revealed that the primordia of the large conical papillae start to develop during the differentiation stage. The primordia of the small conical papillae and conical papillae of the lingual papillae start to develop during the growth stage. At the end of the growth stage, only large conical papillae, three pairs of small conical papillae, and conical papillae of the lingual prominence have similar arrangement as in an adult bird. The shape and arrangement of the remaining small conical papillae probably will be changed after hatching. During embryonic period, the filiform papillae and hair-like papillae are not formed. The embryonic epithelium that covered the mechanical papillae undergoes transformation leading to the formation of multilayered epithelium. During prehatching stage, epithelium becomes orthokeratinized epithelium. In conclusion, the tongue of the domestic goose after hatching is well prepared only for grazing. The filtration of food from water is limited due to the lack of filiform papillae.

Morphofunctional study of the tongue in the domestic duck (Anas platyrhynchos f. domestica, Anatidae): LM and SEM study.

The domestic duck, as a representative of birds living in the water, is considered as a specialist filter-feeder. Behavioral observations of foraging revealed that these birds also use a terrestrial feeding mechanism such as grazing and pecking. This study examined the entirety of the lingual mucosa in relation to the structural adaptations required for this range of feeding activities. The structures on the lateral surfaces of the tongue, the conical and filiform papillae, constitute the food filtration apparatus. The process of pecking involves the spatula-shaped apex of the tongue and a specific horny plate-the lingual nail. In the grazing mechanism, large conical papillae and lamellae in the beak are required. Structures engaged in intra-oral transport include the median groove, lingual combs, the rostral border of the lingual prominence and distinct rows of conical papillae on the lingual prominence. Two types of keratinized epithelia, the ortho- and parakeratinized epithelium, as well as nonkeratinized epithelium cover individual areas of the tongue. The rostral and caudal lingual glands present in the lamina propria of the body, lingual prominence and root of the tongue produce mucus. The specific arrangement of Grandry and Herbst corpuscles form so-called bill-tongue organ monitoring food transportation. Our research confirm that the lingual mucosa in domestic duck is characterized by microstructural species-specific modifications of particular areas of the tongue, which is formed not only under the influence of the filtering mechanism, but also by terrestrial feeding mechanisms such as grazing or pecking.