Anna Pecio

Sexual behaviour of the Montandon's newt, Triturus montandoni (Boulenger) (Caudata: Salamandridae)

In its general pattern the sexual behaviour of Triturus montandoni most resembles that of Triturus helveticus. The courtship consists of three phases: orientation, static display and retreat display, followed by a spermatophore transfer phase. During display the male performs three tail movements: the fan, the whip, and the wave. The relative frequencies of tail movements and the duration of fanning bouts are the main difference between T montandoni, T. helveticus, and T. vulgaris. The phylogenetic relationships between these three species are discussed.

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.

Differentiation and Development of Gonads in the Yellow-Bellied Toad, Bombina variegata L., 1758 (Amphibia: Anura: Bombinatoridae)

The aim of this study was to investigate consecutive stages of gonadal development of the yellow-bellied toad (Bombina variegata) with particular emphasis on the origin of somatic and germ cell lineages as well as the timing of gonial cell migration. Changes in gonadal basal lamina distribution helped to explain the exceptional mode of gonadal differentiation in this species. Atypical and rapid differentiation of the male gonad in B. variegata is the result of the ability of gonial cells to migrate into the center of the gonad relatively early. Thus, the testis medulla contains germ cells from the onset of gonadal differentiation into cortex and medulla, whereas in other anurans a sterile medulla is characteristic of both future testes and ovaries; germ cells translocate into the medulla during the subsequent stage of testis development. This atypical testiculogenesis is probably the result of an acceleration of the sex determination period, indicating a contribution of sex determination heterochrony to the course of gonadogenesis. The results also suggest that medullar cells are derived from proliferating coelomic epithelial cells. Moreover, Sertoli cells constitute an integral part of the germinal epithelium in B. variegata, as in other vertebrates. Spermatids do not contact Sertoli cells just before spermiation and do not form bundles.

Differential effects of testosterone and 17β-estradiol on gonadal development in five anuran species.

Sex hormones are essential for sexual differentiation and play a key role in the development of gonads in amphibians. The goal of this study was to evaluate the influence of exogenous sex steroids, testosterone, and 17β-estradiol (E(2)) on development of gonads in five anuran species differing in their evolutionary positions, sex determination, and mode of gonadogenesis. We found that in two closely related species of fire-bellied toad, Bombina bombina and Bombina variegata, testosterone and E(2) exposure results in sex reversal as well as intersex and undifferentiated gonads. Similarly, sex reversal was observed in Hyla arborea after exposure to male or female sex steroids. Xenopus laevis was sensitive to E(2) but only moderately to testosterone. In Bufo viridis, treatment with either sex hormone provoked a developmental delay in gonads and Bidders organs. Therefore, susceptibility to hormonal sex reversal appeared species dependent but unrelated to genetic sex determination and the type of gonadogenesis. We also found that the onset of sex steroid exposure influences gonad differentiation and the meiotic status of the germ cells depends on their location within the gonad. Our findings reveal differential sensitivity of amphibians to testosterone and E(2), establishing a hierarchy of sensitivity to these hormones among different anuran species.

Retinoic acid homeostasis regulates meiotic entry in developing anuran gonads and in Bidder’s organ through Raldh2 and Cyp26b1 proteins

The vitamin A (retinol) and its metabolites such as retinoic acid (RA) affect vertebrate gametogenesis. The level of RA in cells relies on the balance between its synthesis and degradation. The sex-dependent equilibrium is reached in different ways in various species. It is known that RA induces meiosis in developing gonads in mouse, chicken and urodel amphibians, but its role in anuran amphibians has not been studied. Here we show in six anuran species (Xenopus laevis, Bombina bombina, Hyla arborea, Bufo viridis, Rana arvalis and Rana temporaria) that cultured undifferentiated gonads were insensitive to RA treatment, but the RA induced ectopic meiosis in cultured larval testes. In larval testes of all studied species, the exogenous RA induced leptotene phase of I meiotic prophase in gonia, but only in H. arborea and B. viridis gonia progressed to zygotene phase. In the cultured developing ovaries, exogenous RA led to increase in the number of oocytes as compared to the control. Inhibition of either RA synthesis or RA-receptors prevented meiotic entry in larval gonads of all species. Exogenous RA rescued this inhibitory effect demonstrating that the balance in RA homeostasis plays a key role in meiotic entry in anuran gonads. The localization of two enzymes, Raldh2 and Cyp26b1, which antagonistically control RA levels and whose abundance suggests the sites of RA synthesis and degradation respectively, showed two distinct expression patterns specific for (i) X. laevis, H. arborea, R. arvalis, R. temporaria and (ii) B. bombina, B. viridis. Thus, RA, in correlation with specific expression patterns of Raldh2 and Cyp26b, induces meiosis during gonad development in anurans. In addition, in B. viridis, RA signalling seems important for development of the Bidders organ containing oocytes both in males and females.

Sexual dimorphism of AMH, DMRT1 and RSPO1 localization in the developing gonads of six anuran species

In vertebrates, several genes which are differentially expressed in various species, have been implicated in sex determination and gonadal differentiation. We used immunolocalization to study the expression pattern of three proteins AMH, DMRT1, RSPO1 involved in the sexual differentiation of gonads. The pattern of AMH, DMRT1 and RSPO1 expression was analyzed in X. laevis and in five other divergent anuran species: Bombina bombina, Bufo viridis, Hyla arborea, Rana arvalis and Rana temporaria during gonadal development. The pattern of expression of AMH in the developing testes of six studied anuran species was similar to that described for other vertebrates. AMH was strongly expressed in differentiating Sertoli cells. Interestingly, in B. viridis, R. arvalis and R. temporaria, AMH was also expressed in ovaries. In all studied species, DMRT1 was highly expressed in the developing testes, in both the somatic and germ cells. It was also expressed at low level in ovaries in all studied species, with the exception of H. arborea. RSPO1 was expressed in the developing ovaries, especially in the somatic cells, and was almost undetectable in developing testes in all examined anurans. These developmental expression patterns strongly suggest an involvement of AMH and DMRT1 in the development of male gonads and of RSPO1 in the female gonads. The differences in the expression patterns of these proteins in the gonads of different species might reflect the diversity of gonadal development patterns in anurans resulting from long lasting and diverged paths of their evolution.

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.

Ultrastructure of the epithelial cells in the aspermatogenic part of the testis in Mimagoniates barberi (Teleostei: Characidae: Glandulocaudinae) and the role of their secretions in spermatozeugmata formation

Two types of unencapsulated sperm bundles are present in the milt of the internally inseminating characid fish, Mimagoniates barberi. Our ultrastructural study revealed that sperm bundles of both types are formed within the efferent ducts of the posterior, aspermatogenic part of the testis. The epithelial cells of the testicular tubules of the aspermatogenic portion of M. barberi testis show signs of both secretory and phagocytic activity. Rough endoplasmic reticulum, polyribosomes, Golgi complexes, secretory vesicles and vacuoles abound in the cytoplasm. Adluminal surface of the epithelial cells often exhibits extensions of various form. Two types of secretions are present in the efferent ducts. In more proximal ducts the secretion is finely granular, whereas in more distal ducts groups of coalescing spermatozoa are accompanied by a fibrous secretion. The sperm are aligned along strings of this secretion which is most probably instrumental in sperm clumping. In the most caudal portion of the testis where only fully formed sperm bundles of both types are present no structured secretion in the lumen of the efferent ducts is found. No secretion between the sperm within the bundles is visible. The results of histochemical staining indicate that the secretion in the tubules has no carbohydrate component and is proteinaceous.

Differential effects of busulfan on gonadal development in five divergent anuran species

The aim of this paper was to investigate the effects of germ cell depletion on the sexual differentiation of gonads in five anuran species. We used busulfan to eliminate the germ cells. Our results indicate that germ cells are not required for gonadal ridge formation or the development of the undifferentiated gonads. We observed a gradual degeneration of gonads in studied species and the transdifferentiation of the whole gonads into large fat bodies in Xenopus laevis. In the latter the sexual differentiation of gonads or seminiferous tubules were not impaired in the absence of germ cells. Thus, the X. laevis may serve as a model to study the human Del Castillo syndrome. Our study shows that in anuran amphibians the germ cells are not necessary for the formation of the testis, but they are crucial for development of the ovaries and are required for the maintenance of the gonadal structure.

Sperm and Spermatozeugma Structure in Xenurobrycon (Teleostei: Characidae: Stevardiinae: Xenurobryconini)

Abstract The main purpose of this study was to describe the morphology of the sperm packets produced by species in the genus Xenurobrycon. Males of the three species analyzed, X. macropus, X. polyancistrus, and X. heterodon, produce unencapsulated sperm bundles (spermatozeugmata) that are essentially identical in morphology. In longitudinal histological sections, each spermatozeugma is feather-like, with the tips of the sperm heads associated with a core that stains with the periodic acid-Schiff reagent (PAS+). As revealed by SEM, sperm heads are exposed only at the anterior end of the packet. Flagella cover the remainder of the packet and form a “tail” at the posterior end. The flagellum probably exits from a cytoplasmic collar at one side of the flattened sperm head, which tapers posteriorly. Spermatozeugmata are formed within the spermatocysts and only complete packets are released into the sperm ducts. Very few loose spermatozoa are seen within these ducts, attesting to the high degree of compactness of these sperm bundles. Insemination is confirmed in a specimen of X. macropus, whose ovary contained spermatozeugmata, demonstrating that intact packets can be transferred to the ovary by the male. A lateral flagellum, which is present in the xenurobryconin genera Scopaeocharax, Tyttocharax, and Xenurobrycon, may prove to be a synapomorphy for the entire tribe Xenurobryconini. In addition to providing information on reproductive morphology in these species, the results also demonstrate the potential use of characters obtained from such microscopic analyses in phylogenetic studies.