Evolutionary history of mental glands in turtles reveals a single origin in an aquatic ancestor and recurrent losses independent of macrohabitat.

Despite the relevance of chemical communication in vertebrates, comparative examinations of macroevolutionary trends in chemical signaling systems are scarce. Many turtle and tortoise species are reliant on chemical signals to communicate in aquatic and terrestrial macrohabitats, and many of these species possess specialized integumentary organs, termed mental glands (MGs), involved in the production of chemosignals. We inferred the evolutionary history of MGs and tested the impact of macrohabitat on their evolution. Inference of ancestral states along a time-calibrated phylogeny revealed a single origin in the ancestor of the subclade Testudinoidea. Thus, MGs represent homologous structures in all descending lineages. We also inferred multiple independent losses of MGs in both terrestrial and aquatic clades. Although MGs first appeared in an aquatic turtle (the testudinoid ancestor), macrohabitat seems to have had little effect on MG presence or absence in descendants. Instead, we find clade-specific evolutionary trends, with some clades showing increased gland size and morphological complexity, whereas others exhibiting reduction or MG loss. In sister clades inhabiting similar ecological niches, contrasting patterns (loss vs. maintenance) may occur. We conclude that the multiple losses of MGs in turtle clades have not been influenced by macrohabitat and that other factors have affected MG evolution.

The chemistry and histology of sexually dimorphic mental glands in the freshwater turtle, Mauremys leprosa.

Despite evidence from anatomy, behavior and genomics indicating that the sense of smell in turtles is important, our understanding of chemical communication in this group is still rudimentary. Our aim was to describe the microanatomy of mental glands (MGs) in a freshwater turtle, Mauremys leprosa (Geoemydidae), and to assess the chemical composition of their secretions with respect to variation among individuals and between sexes. MGs are paired sac-like organs on the gular region of the neck and are dimorphic in this species with males having fully functional holocrine glands while those of females appear non-secretory and vestigial. In adult males, the glandular epithelium of the inner portion of the gland provides exocytotic products as well as cellular debris into the lumen of the gland. The contents of the lumen can be secreted through the narrow duct portion of the gland ending in an orifice on the surface of the skin. Females have invaginated structures similar in general outline to male glands, but lack a glandular epithelium. Using gas chromatography coupled to mass spectrometry, we identified a total of 61 compounds in mental gland secretions, the most numerous being carboxylic acids, carbohydrates, alkanes, steroids and alcohols. The number of compounds per individual varied widely (mean (median) ± SD = 14.54 (13) ± 8.44; min = 3; max = 40), but only cholesterol was found in all samples. We found that the relative abundances of only six chemicals were different between the sexes, although males tended to have larger amounts of particular compounds. Although the lipid fraction of mental gland secretions is rich in chemical compounds, most occur in both sexes suggesting that they are metabolic byproducts with no role in chemical signaling. However, the relative amounts of some compounds tended to be higher in males, with significantly larger amounts of two carboxylic acids and one steroid, suggesting their putative involvement in chemical communication.

N-Cadherin Is Critical for the Survival of Germ Cells, the Formation of Steroidogenic Cells, and the Architecture of Developing Mouse Gonads.

Normal gonad development assures the fertility of the individual. The properly functioning gonads must contain a sufficient number of the viable germ cells, possess a correct architecture and tissue structure, and assure the proper hormonal regulation. This is achieved by the interplay between the germ cells and different types of somatic cells. N-cadherin coded by the Cdh2 gene plays a critical role in this interplay. To gain an insight into the role of N-cadherin in the development of mouse gonads, we used the Cre-loxP system to knock out N-cadherin separately in two cell lines: the SF1+ somatic cells and the OCT4+ germ cells. We observed that N-cadherin plays a key role in the survival of both female and male germ cells. However, the N-cadherin is not necessary for the differentiation of the Sertoli cells or the initiation of the formation of testis cords or ovigerous cords. In the later stages of gonad development, N-cadherin is important for the maintenance of testis cord structure and is required for the formation of steroidogenic cells. In the ovaries, N-cadherin is necessary for the formation of the ovarian follicles. These results indicate that N-cadherin plays a major role in gonad differentiation, structuralization, and function.

Tissue-specific knockout of E-cadherin (Cdh1) in developing mouse gonads causes germ cells loss.

The normal course of gonad development is critical for the sexual development and reproductive capacity of the individual. During development, an incipient bipotential gonad which consists of unorganized aggregate of cells, must differentiate into highly structured testis or ovary. Cell adhesion molecules (CAMs) are a group of proteins crucial for segregation and aggregation of different cell types to form different tissues. E-cadherin (Cdh1) is one of the CAMs expressed in the developing gonads. We used tissue-specific knockout of Cdh1 gene in OCT4+ germ cells and, separately, in SF1+ somatic cells of developing gonads. The knockout of E-cadherin in somatic cells caused decrease in the number of germ cells, while the knockout in the germ cells caused their almost complete loss. Thus, the presence of E-cadherin in both the germ and somatic cells is necessary for the survival of germ cells. Although the lack of E-cadherin did not impair cell proliferation, it enhanced apoptosis, which was a possible cause of germ cell loss. However, the somatic cells of the gonad differentiated normally into Sertoli cells in the testis cords, and into follicular cells in the ovaries. The testis and ovigerous cords maintained their integrity; they were covered by continuous basement membranes. The testicular interstitium with steroidogenic fetal Leydig cells did not show any noticeable changes. However, in the female gonads, because of the lack of germ cells, the ovarian follicles were absent. The sex determination and sexual differentiation of the gonad were not impaired. These results underscore an important role of E-cadherin in germ cell survival and gonad development.

Expression of primary cilia-related genes in developing mouse gonads.

Mechanisms governing differentiation of the bipotential gonad into the testes or ovaries are complex and still vague. The primary cilium is an organelle involved in cell signaling, which controls the development of many organs, but the role of primary cilium in the sex determination and sexual differentiation of gonads is com-pletely unknown. Here we studied the expression of genes involved in primary cilium formation and function-ing in fetal mouse gonads, before, during and after sexual differentiation. We studied the expression of 175 primary cilia-related genes using microarray technique. 144 of these genes were ubiquitously expressed in all studied cell types with no significant differences in expression level. Such a high level of expression of primary cilia-related genes in developing mouse gonads suggests that the primary cilia and/or primary cilia-related genes are important for the development of both somatic and germline component of the gonads. Only 31 genes showed a difference in expression between different cell types, which suggests that they have different functions in the somatic and germ cells. These results justify further studies on the role of primary cilia and the primary cilia-related genes in gonad development.

Transcriptional profiling validates involvement of extracellular matrix and proteinases genes in mouse gonad development.

Extracellular matrix (ECM) plays an important scaffolding role in the establishment of organs structure during development. A great number of ECM components and enzymes (proteinases) regulating formation/degradation of ECM during organ remodeling have been identified. In order to study the role of ECM in the mouse gonad development, especially during sexual differentiation of the gonads when the structure of the testis and ovary becomes established, we performed a global analysis of transcriptome in three main cell types of developing gonad (supporting, interstitial/stromal and germ cells) using transgenic mice, cell sorting and microarray. The genes coding for ECM components were mostly expressed in two gonadal cell lines: supporting and interstitial/stromal cells. These two cell lines differed in the expression pattern of ECM components, which suggests that ECM components might be crucial for differentiation of gonad compartments (for example testis cords vs. interstitium in XY gonads). Collagens and proteoglycans coding genes were mainly expressed in the interstitium/stromal cells, while non-collagen glycoproteins and matricellular coding genes were expressed in both cell lines. We also analyzed the expression of genes encoding ECM enzymes that are secreted to the ECM where they remodel the scaffolding of developing organs. We found that the ECM enzyme genes were also mostly expressed in supporting and interstitial/stromal cells. In contrast to the somatic cells, the germ cells expressed only limited number of ECM components and enzymes. This suggests that the germ line cells do not participate, or play only a minor role, in the sculpting of the gonad structure via ECM synthesis and remodeling. Importantly, the supporting cells showed the sex-specific pattern of expression of ECM components. However, the pattern of expression of most ECM enzymes in the somatic and germ cells is independent on the sex of the gonad. Further studies are required to elucidate the exact roles of identified genes in sexual differentiation of the gonads.

Cell adhesion molecules expression pattern indicates that somatic cells arbitrate gonadal sex of differentiating bipotential fetal mouse gonad.

Unlike other organ anlagens, the primordial gonad is sexually bipotential in all animals. In mouse, the bipotential gonad differentiates into testis or ovary depending on the genetic sex (XY or XX) of the fetus. During gonad development cells segregate, depending on genetic sex, into distinct compartments: testis cords and interstitium form in XY gonad, and germ cell cysts and stroma in XX gonad. However, our knowledge of mechanisms governing gonadal sex differentiation remains very vague. Because it is known that adhesion molecules (CAMs) play a key role in organogenesis, we suspected that diversified expression of CAMs should also play a crucial role in gonad development. Using microarray analysis we identified 129 CAMs and factors regulating cell adhesion during sexual differentiation of mouse gonad. To identify genes expressed differentially in three cell lines in XY and XX gonads: i) supporting (Sertoli or follicular cells), ii) interstitial or stromal cells, and iii) germ cells, we used transgenic mice expressing EGFP reporter gene and FACS cell sorting. Although a large number of CAMs expressed ubiquitously, expression of certain genes was cell line- and genetic sex-specific. The sets of CAMs differentially expressed in supporting versus interstitial/stromal cells may be responsible for segregation of these two cell lines during gonadal development. There was also a significant difference in CAMs expression pattern between XY supporting (Sertoli) and XX supporting (follicular) cells but not between XY and XX germ cells. This indicates that differential CAMs expression pattern in the somatic cells but not in the germ line arbitrates structural organization of gonadal anlagen into testis or ovary.

Gonads in Histiostoma mites (Acariformes: Astigmata): structure and development.

The development of male and female gonads in arrhenotokous and thelytokous species of Histiostoma was studied using transmission electron microscopy (TEM). All instars were examined: larvae, protonymphs, facultative heteromorphic deutonymphs (=hypopi), tritonymphs, and adults. In testis primordium, spermatogonia surrounding a testicular central cell (TCC) with a gradually enlarging, branched nucleus are present already at the larval stage. Spermatogonia and the TCC are connected via narrow, tubular intercellular bridges revealing that the TCC is a germline cell. Spermatocytes appear at the protonymphal stage. At the heteromorphic deutonymph stage, the testis primordium is similar to that of the protonymph, but in the tritonymph it is much larger and composed as in the adult: spermatids as well as sperm cells are present. The latter are congregated ventrally in the testis at the entrance of the deferent duct. In the larval ovary, an eccentrically located ovarian nutritive cell (ONC) is surrounded by oogonia which are connected with the ONC via tubular intercellular bridges. In later stages, the ovary grows and oocytes appear in the protonymph. Meiotic synaptonemal complexes in oocytes occur from the tritonymph stage. At about the time of the final molting, tubular intercellular bridges transform into peculiar diaphragm-crossed bridges known only in Histiostoma mites. In the adult female, growing oocytes at the end of previtellogenesis lose intercellular bridges and move ventro-laterally to the ovarian periphery towards the oviduct entrance. Vitellogenesis occurs in oviducts. Germinal cells in both the testis and ovary are embedded in a few somatic stroma cells which may be well discernible already in the larval ovary; in the testis, somatic stroma cells are evident not earlier than the end of the tritonymphal stage. The ovary has a thin wall of flat somatic cells, whereas the testis is covered by a basal lamina only. The obtained results suggest that gonads in Histiostoma and other Astigmata originate from two primordial cells only.

HGF/SF increases number of skin melanocytes but does not alter quality or quantity of follicular melanogenesis.

Melanins are an important factor determining the vulnerability of mammalian skin to UV radiation and thus to UV-induced skin cancers. Transgenic mice overexpressing hepatocyte growth factor/scatter factor (HGF/SF) have extra-follicular dermal melanocytes, notably in the papillary upper dermis, and are susceptible to UV-induced melanoma. Pigmented HGF/SF neonatal mice are more susceptible than albino HGF/SF animals to UVA -induced melanoma, indicating an involvement of melanin in melanoma formation. This raises the question of the effect of transgenic HGF/SF on melanization. We developed a methodology to accurately quantitate both the production of melanin and the efficiency of melanogenesis in normal, and HGF/SF transgenic mice in vivo. Skin and hair shafts of 5 day old and adult (3 week old) C57BL/6-HGF/SF and corresponding C57BL/6 wild type mice were investigated by electron paramagnetic resonance spectroscopy (EPR) to quantitate melanin, by transmission electron microscopy (TEM) for the presence of melanosomes, and by standard histology and by Western blotting and zymography to determine the expression and activity of melanogenesis-related proteins. Eumelanin but no phaeomelanin was detected in transgenic C57BL/6-HGF and C57BL/6 wild type mice. Transgenic HGF/SF overexpression did not change the type of melanin produced in the skin or hair, did not affect the terminal content of melanin production in standard samples of hair and did not influence hair cycle/morphogenesis-related changes in skin thickness. No melanocytes were found in the epidermis and no melanosomes were found in epidermal keratinocytes. HGF/SF transgenic mice thus lack the epidermal melanin UV-protection found in constitutively dark human skin. We conclude that melanocytes in the HGF/SF transgenic mouse, particularly in the papillary dermis, are vulnerable to UVA which interacts with eumelanin but not phaeomelanin to induce melanoma.

Pheomelanin in the skin of Hymenochirus boettgeri (Amphibia: Anura: Pipidae).

Pheomelanin is supposed to be the first type of melanin found in vertebrates, in contrast to the main type - eumelanin. Our study aimed at detecting pheomelanin in the skin of Hymenochirus boettgerii. We employed electron paramagnetic resonance (EPR) spectroscopy, and transmission electron microscopy (TEM), supplemented with standard histology and immunochemistry. We identified pheomelanin in the dorsal skin of adult frogs (not only in the dermis, but also in the epidermis) and in the dorsal tadpole. Our work identifies Hymenochirus boettgerii as a model in the basic study on the mechanism, evolution and role of melanogenesis in animals, including human.

Morphology of the air-breathing stomach of the catfish Hypostomus plecostomus.

Histological and ultrastructural investigations of the stomach of the catfish Hypostomus plecostomus show that its structure is different from that typical of the stomachs of other teleostean fishes: the wall is thin and transparent, while the mucosal layer is smooth and devoid of folds. The epithelium lining the whole internal surface of the stomach consists of several types of cells, the most prominent being flattened respiratory epithelial cells. There are also two types of gastric gland cells, three types of endocrine cells (EC), and basal cells. The epithelial layer is underlain by capillaries of a diameter ranging from 6.1-13.1 microm. Capillaries are more numerous in the anterior part of the stomach, where the mean number of capillary sections per 100 microm of epithelium length is 4, compared with 3 in the posterior part. The cytoplasm of the epithelial cells, apart from its typical organelles, contains electron-dense and lamellar bodies at different stages of maturation, which form the sites of accumulation of surfactant. Small, electron-dense vesicles containing acidic mucopolysaccharides are found in the apical parts of some respiratory epithelial cells. Numerous gastric glands (2 glands per 100 microm of epithelium length), composed of two types of pyramidal cells, extend from the surface epithelium into the subjacent lamina propria. The gland outlets, as well as the apical cytoplasm of the cells are Alcian blue-positive, indicating the presence of acidic mucopolysaccharides. Zymogen granules have not been found, but the apical parts of cells contain vesicles of variable electron density. The cytoplasm of the gastric gland cells also contains numerous electron-dense and lamellar bodies. Gastric gland cells with electron-dense cytoplasm and tubulovesicular system are probably involved in the production of hydrochloric acid. Fixation with tannic acid as well as with ruthenium red revealed a thin layer of phospholipids and glycosaminoglycans covering the entire inner surface of the stomach. In regions of the epithelium where the capillaries are covered by the thin cytoplasmic sheets of the respiratory epithelial cells, a thin air-blood barrier (0.25-2.02 microm) is formed, thus enabling gaseous exchange. Relatively numerous pores closed by diaphragms are seen in the endothelium lining the apical and lateral parts of the capillaries. Between gastric gland cells, solitary, noninnervated endocrine cells (EC) of three types were found. EC are characterized by lighter cytoplasm than the surrounding cells and they contain dense core vesicles (DCV) with a halo between the electron-dense core and the limiting membrane. EC of type I are the most abundant. They are of an open type, reaching the stomach lumen. The round DCV of this type, with a diameter from 92-194 nm, have a centrally located core surrounded by a narrow halo. EC of type II are rarely observed and are of a closed type. They possess two kinds of DCV with a very narrow halo. The majority of them are round, with a diameter ranging from 88-177 nm, while elongated ones, 159-389 nm long, are rare. EC of type III are numerous and also closed. The whole cytoplasm is filled with large DCV: round, with a diameter from 123-283 nm, and oval, 230-371 nm long, both with a core of irregular shape and a wide, irregular halo. EC are involved in the regulation of digestion and probably local gas exchange. In conclusion, the thin-walled stomach of Hypostomus plecostomus, with its rich network of capillaries, has a morphology suggesting it is an efficient organ for air breathing.

Adaptations to the air breathing in the posterior intestine of the catfish (Corydoras aeneus, Callichthyidae). A histological and ultrastructural study.

A light and transmission electron microscopic study of the intestine of catfish C. aeneus shows that the anterior part of the intestine is a site of digestion and absorption and its structure is typical of that of other teleostean fishes. However, in this species the thin-walled posterior intestine is adapted to air breathing. In this region mucosa is smooth and lined with respiratory epithelium with capillary network. Several types of cells are observed in the epithelium: flattened respiratory epithelial cells with short microvili, goblet cells, scarce epithelial cells with numerous longer microvilli, and two types of endocrine cells (EC). The solitary brush cells with several long and thick microvilli described here are the first observation of such cells in the gastrointestinal tract of fishes. Bodies of respiratory epithelial cells lie between capillaries. Their cytoplasm, apart from typical organelles contains dense and lamellar bodies, which are a site of accumulation of surfactant. In regions where capillaries are covered by thin cytoplasmic sheets of respiratory epithelial cells, a thin (0.24-3.00 microm) air-blood barrier is formed, thus enabling gas exchange. Epithelial cells with longer microvilli do not participate in the formation of the air-blood barrier and are probably responsible for absorbtion. EC of the closed type are dispersed within the epithelium. Their cytoplasm contains characteristic round or oval dense core vesicles 69 to 230 nm in diameter. The role of EC and brush cells in the regulation of processes related to absorbtion, and to respiration, is disscused.