Thermal and oxygen conditions during development cause common rough woodlice (Porcellio scaber) to alter the size of their gas-exchange organs.

Terrestrial isopods have evolved pleopodal lungs that provide access to the rich aerial supply of oxygen. However, isopods occupy conditions with wide and unpredictable thermal and oxygen gradients, suggesting that they might have evolved adaptive developmental plasticity in their respiratory organs to help meet metabolic demand over a wide range of oxygen conditions. To explore this plasticity, we conducted an experiment in which we reared common rough woodlice (Porcellio scaber) from eggs to maturation at different temperatures (15 and 22 °C) combined with different oxygen levels (10% and 22% O2). We sampled animals during development (only females) and then examined mature adults (both sexes). We compared woodlice between treatments with respect to the area of their pleopod exopodites (our proxy of lung size) and the shape of Bertalanffy's equations (our proxy of individual growth curves). Generally, males exhibited larger lungs than females relative to body size. Woodlice also grew relatively fast but achieved a decreased asymptotic body mass in response to warm conditions; the oxygen did not affect growth. Under hypoxia, growing females developed larger lungs compared to under normoxia, but only in the late stage of development. Among mature animals, this effect was present only in males. Woodlice reared under warm conditions had relatively small lungs, in both developing females (the effect was increased in relatively large females) and among mature males and females. Our results demonstrated that woodlice exhibit phenotypic plasticity in their lung size. We suggest that this plasticity helps woodlice equilibrate their gas exchange capacity to differences in the oxygen supply and metabolic demand along environmental temperature and oxygen gradients. The complex pattern of plasticity might indicate the effects of a balance between water conservation and oxygen uptake, which would be especially pronounced in mature females that need to generate an aqueous environment inside their brood pouch.

Spermatogenesis in the inseminating African butterflyfish Pantodon buchholzi (Teleostei: Osteoglossiformes: Pantodontidae) with the revision of residual bodies formation.

The aim of this study was to analyse spermatogenesis in the African butterflyfish, Pantodon buchholzi, using transmission electron microscopy and scanning electron microscopy. P. buchholzi is the most basal teleost that exhibits insemination and produces a highly complex introsperm with the most elongate midpiece known in teleost fishes. Their early stages (spermatogonia and spermatocytes) do not differ greatly from those of other fishes, with the exception of Golgi apparatus degradation appearing as spindle-shaped bodies (SSBs). In round, early spermatids, the development of the flagellum begins after the migration of the centriolar complex towards the nucleus. Later, the elongation of the midpiece coincides with the displacement of the mitochondria and their fusion to produce nine mitochondrial derivatives (MDs). In these spermatids, the nucleus is situated laterally to the midpiece, with condensing chromatin in the centre of the nucleus. Within the midpiece, the flagellum is located within a cytoplasmic canal and is surrounded by a cytoplasmic sleeve containing fibres, MDs and a great amount of cytoplasm located on one side. During the next phase, nuclear rotation, the highly condensed chromatin is displaced to a position above the centriolar apparatus, whereas chromatin-free nucleoplasm is transferred to the cytoplasm. Later, this nucleoplasm, still surrounded by the nuclear membrane, is eliminated into the cyst lumen as the nucleoplasmic packet. Within the highly elongate spermatids, other excess organelles (SSBs, endoplasmic reticulum and mitochondria) are eliminated as residual bodies (RBs). Fully developed spermatozoa, which contain conical-shaped nuclei, eventually coalesce to form unencapsulated sperm packets (spermatozeugmata) that are surrounded by RBs at the level of the extremely elongate midpieces. Later, RBs are removed at the periphery of the cyst by means of phagocytosis by Sertoli cells.

A new type of spermiogenesis in teleost fish: Formation of the aflagellate sperm in Campylomormyrus compressirostris (Osteoglossomorpha: Mormyridae).

Osteoglossomorpha, the bony tongue fishes, show great variation in morphology, behavioural strategies, reproductive biology and gamete ultrastructure. The order Osteoglossiformes is the only vertebrate taxon, in which four types of sperm (monoflagellate, biflagellate and aflagellate aquasperm and the complex introsperm) have been described. It is also the only vertebrate lineage in which aflagellate spermatozoa exist. The aim of this study was to analyse the structure of the testis and the process of spermiogenesis in the mormyrid Campylomormyrus compressirostris during the breeding season using light and electron microscopy (transmission and scanning). Males of this species have a single testis of the anastomosing tubular type. The tubules of the anterior part of the testis contain cysts with developing germ cells, and this region is much wider than the posterior part, which consists of efferent ducts filled with sperm cells. The cysts are filled with single or mitotic spermatogonia, primary and secondary spermatocytes and early spermatids. At the stage of spermatids with fine granular chromatin, the cysts rupture and successive stages of spermatid differentiation take place in the testicular lumen; we therefore characterise this process as 'extracystic spermiogenesis'. Sperm development in C. compressirostris is extremely simple and involves chromatin condensation in the central region of the nucleus, a slight decrease in nuclear volume, the appearance of numerous vesicles in the cytoplasm that form a tubular-vesicular system at the base of the nucleus. Both centrioles and mitochondria are translocated to the peripheral region of the midpiece, which forms the opposite pole to the nucleus. There are many differences between the types of spermiogenesis described so far in teleosts and that found in C. compressirostris, including the loss of flagellum formation. This unique type of spermiogenesis is restricted to species of the families Mormyridae and Gymnarchidae, all of which possess aflagellate spermatozoa. Our data demonstrate that the spermatid differentiation and existence of the aflagellate spermatozoon are a unique phenomena not only among teleosts but also in the whole vertebrate lineage.

Differential effects of testosterone and 17ß-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 17ß-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 Bidder's 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.

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.

Roundup in the Reproduction of Crucian Carp (Carassius carassius): An In Vitro Effect on the Pituitary Gland and Ovary.

Roundup, the most popular herbicide in global agriculture, is regarded as an endocrine disruptor causing alterations of important hormones at the hypothalamic-pituitary-gonadal axis as well as impairment of gametogenesis. The whole pituitary glands of crucian carp (Carassius carassius) were incubated for 3 h in the medium containing Roundup (0-control, 1 and 10 ng/mL). The level of luteinizing hormone (LH), and mRNA transcript abundance of kisspeptin (kiss-1) and its receptor (gpr54), were determined. The isolated ovarian fragments were incubated for 24 h in the presence of Roundup and the following effects on reproductive parameters were determined: the final oocyte maturation and ovulation, structural changes in follicles, secretion of 17,20ß-progesterone (17,20ß-P) as well as mRNA transcript abundance of the luteinizing hormone receptor (lhr), estrogen receptors (erα, erß1, erß2), and zona radiata (chorion) proteins (zp2 and zp3). Roundup inhibited final oocyte maturation and decreased the percentage of ovulated eggs, and furthermore, caused structural changes in the ovarian follicular components. There were no significant changes in the measured hormone levels and analyzed genes mRNA transcript abundance. Summing up, obtained results indicate that Roundup may adversely affect oocyte maturation and the quality of eggs, suggesting that exposure to this herbicide can lead to reproductive disorders in fish.

Diversity of Balbiani body formation in internally and externally fertilizing representatives of Osteoglossiformes (Teleostei: Osteoglossomorpha).

During the early stages of oogenesis, the Balbiani body is formed in the primary oocytes. It consists of the Golgi apparatus, endoplasmic reticulum (ER), and numerous mitochondria aggregated with germ plasm, but its form may differ among animals. Hypothetically, during oogenesis oocytes become adapted to future development in two different environments depending on internal or external fertilization. We aimed to investigate, using light and transmission electron microscopy, the development of the Balbiani body during oogenesis in representatives of Osteoglossiformes, one of the most basal Teleostei groups. We analyzed the structure of oogonia and primary oocytes in the internally fertilizing butterflyfish Pantodon buchholzi and the externally fertilizing Osteoglossum bicirrhosum and Arapaima gigas to compare formation of the Balbiani body in relation to modes of fertilization. We demonstrated that the presence of the germ plasm as well as the fusion and fission of mitochondria are the conserved features of the Bb. However, each species exhibited also some peculiar features, including the presence of three types of ooplasm with different electron density and mitochondria-associated membranes in P. buchholzi; annulate lamellae, complexes of the Golgi apparatus, ER network, and lysosome-like bodies in O. bicirrhosum; as well as karmellae and whorls formed by the lamellae of the ER in A. gigas. Moreover, the form of the germ plasm observed in close contact with mitochondria differed between osteoglossiforms, with a "net-like" structure in P. buchholzi, the presence of numerous strings in O. bicirrhosum, and irregular accumulations in A. gigas. These unique features indicate that the extreme diversity of gamete structure observed so far only in the spermatozoa of osteoglossiforms is also characteristic for oocyte development in these basal teleosts. Possible reason of this variability is a period of about 150 million years of independent evolution of the lineages.

Tilapia Lake Virus-Induced Neuroinflammation in Zebrafish: Microglia Activation and Sickness Behavior.

In mammals, the relationship between the immune system and behavior is widely studied. In fish, however, the knowledge concerning the brain immune response and behavioral changes during brain viral infection is very limited. To further investigate this subject, we used the model of tilapia lake virus (TiLV) infection of zebrafish (Danio rerio), which was previously developed in our laboratory. We demonstrated that TiLV persists in the brain of adult zebrafish for at least 90 days, even when the virus is not detectable in other peripheral organs. The virions were found in the whole brain. During TiLV infection, zebrafish displayed a clear sickness behavior: decreased locomotor activity, reduced food intake, and primarily localizes near the bottom zone of aquaria. Moreover, during swimming, individual fish exhibited also unusual spiral movement patterns. Gene expression study revealed that TiLV induces in the brain of adult fish strong antiviral and inflammatory response and upregulates expression of genes encoding microglia/macrophage markers. Finally, using zebrafish larvae, we showed that TiLV infection induces histopathological abnormalities in the brain and causes activation of the microglia which is manifested by changes in cell shape from a resting ramified state in mock-infected to a highly ameboid active state in TiLV-infected larvae. This is the first study presenting a comprehensive analysis of the brain immune response associated with microglia activation and subsequent sickness behavior during systemic viral infection in zebrafish.

Type I interferon-dependent response of zebrafish larvae during tilapia lake virus (TiLV) infection.

Tilapia lake virus (TiLV; genus: Tilapinevirus, family: Amnoonviridae) is a recently characterised enveloped virus with a linear, negative-sense single-stranded RNA genome, which causes high mortality in tilapia species. In the present study, we demonstrated that zebrafish (Danio rerio) larvae are susceptible to TiLV infection upon systemic injection. TiLV replicated in zebrafish larvae and caused their high mortality (of about 70%). Histopathological examination revealed that TiLV infection caused pathological abnormalities in zebrafish larvae that were well visible within the brain. Moreover, gene expression analysis revealed that TiLV infection induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I (ddx58), tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), and pro-inflammatory cytokine (il-1ß). We also demonstrated the protective role of the recombinant zebrafish IFNϕ1 on the survival of zebrafish larvae during TiLV infection. Our results show the importance of type I IFN response during TiLV infection in zebrafish larvae and demonstrate that zebrafish is a good model organism to study interactions between TiLV - a newly emerging in aquaculture virus, and fish host.

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.

Ultrastructural examination of spermiogenesis and spermatozoon ultrastructure in Congo tetra Phenacogrammus interruptus Boulenger, 1899 (Ostariophysi: Characiformes: Alestidae).

Ultrastructural studies of spermiogenesis in Phenacogrammus interruptus using transmission electron microscopy revealed that the process is characterized by flagellum development, formation of a cytoplasmic canal, nuclear rotation, and nuclear fossa formation. Chromatin compaction proceeds during spermatid transformation within the spermatocysts as well as after spermiation within the lumen of the efferent ducts. The spermatozoon is of primitive type and exhibits characters typical for Type I aquasperm. The head consists of a spherical nucleus with highly condensed chromatin and a centrally located electron lucent area connected to a moderate-sized nuclear fossa. The nuclear fossa contain centrioles in perpendicular arrangement, surrounded by osmiophilic fibrous material. In the short midpiece, several mitochondria and vesicles are unevenly distributed in the cytoplasm forming the cytoplasmic collar at the base of the nucleus. The cytoplasmic collar surrounds the initial part of the flagellum, running in the cytoplasmic canal. The flagellar axoneme has a typical pattern (9 x 2 + 2) and the flagellum contains membranous compartments in the portion immediately posterior to the termination of the cytoplasmic canal.

Evidence of the sterility of allotetraploid Cobitis loaches (Teleostei, Cobitidae) using testes ultrastructure.

The diploid-polyploid populations of Cobitis distributed in Poland are usually composed of the spined loach Cobitis taenia or, less often, the Danubian loach C. elongatoides and their triploid (females) and tetraploid hybrids (females and males). The aim of this study was to determine whether tetraploid males participate in the reproduction process by analyzing their testis ultrastructure and the process of spermatogenesis in comparison with diploid males of both parental species. Tetraploid loaches were obtained from three different diploid-polyploid populations distributed in Poland. The structure of Cobitis testes are typical for most Teleostei fish with cystic-type spermatogenesis. The successive stages of developing germ cells are enclosed within cysts formed by the Sertoli cells. This paper morphologically describes the different germ cell stages of spermatogenesis (spermatogonia, spermatocytes, spermatids, and spermatozoa) of C. taenia and C. elongatoides and provides a pioneering ultrastructural analysis of tetraploid Cobitis testes which reveals their unusual structure for the first time. Thus, cysts with normal spermatogonia and spermatocytes (pachyten or leptoten stages) containing synaptonemal complexes were present and no spermatids or spermatozoa were observed. Moreover, in contrast to previously analyzed diploid species, single cells or all of the cells within the cysts displayed chromatin condensation and/or chromatin fragmentation. The obtained results clearly demonstrated that tetraploid males are sterile and diploids are fertile and are the only sperm donors in the reproduction processes of diploid-polyploid Cobitis populations.

Sperm and spermatozeugma ultrastructure in the inseminating species Tyttocharax cochui, T. tambopatensis, and Scopaeocharax rhinodus (Pisces: Teleostei: Characidae: Glandulocaudinae: Xenurobryconini).

This article presents the scanning and transmission electron microscopy of the spermatozoa and sperm packets of three inseminating species of the glandulocaudine tribe Xenurobryconini. All three species, Scopaeocharax rhinodus, Tyttocharax cochui, and T. tambopatensis produce unencapsulated sperm packets (= spermatozeugmata) of similar morphology. The external anterior surface of each spermatozeugma is comprised of elongate sperm heads arranged in parallel, and the posterior part is made up of tightly packed flagella. The interior of the anterior portion consists of alternating layers of sperm heads and flagella. The remarkable integrity of each packet appears to be maintained through an electron-dense secretion seen among all parts of the cells. Spermatozeugma formation takes place within the spermatocysts at the end of spermiogenesis and at spermiation fully formed packets are released. Morphology of the mature spermatozoa was similar in all three species. Each nucleus is elongate, flattened along most of its length, and tapers at either end. The two centrioles are nearly parallel to one another and are located just anterior to the nucleus. Elongate mitochondria are located along the nucleus. The single flagellum, which lacks axonemal fins, is initially contained within a short cytoplasmic collar. Accessory microtubules run parallel to the long axis of the nucleus just beneath the plasma membrane. During spermiogenesis, no nuclear rotation occurs and the cytoplasmic canal containing the flagellum elongates along with the nucleus. However, prior to spermiation all but the anterior portion of the collar degenerates. The sperm modifications observed in these species are discussed as adaptations to the unique reproductive habit of insemination.

Spermiogenesis and fine structure of the spermatozoon in a headstander, Chilodus punctatus (Teleostei, Characiformes, Anostomidae).

The main characteristic features of spermiogenesis in Chilodus punctatus (Characiformes) are rotation of the nucleus, development of a nuclear fossa, which extends as a narrow invagination deep into the nucleus and the way in which flagellum is formed. The chromatin condensation proceeds during the spermiogenesis from heterogeneous through homogenous and granular to a highly compact one present in the mature spermatozoon. Mature Ch. punctatus spermatozoon shows a spherical nucleus, short midpiece and flagellum with lateral fins. The centrioles are in perpendicular arrangement and are located in the deep nuclear fossa, which extends towards the anterior pole of the nucleus. The midpiece contains a few mitochondria, which are separated from the anterior fragment of flagellum by the cytoplasmic channel. Spermiogenesis and spermatozoon ultrastructure conform to the pattern observed in other ostariophysans, but for the first time the presence of lateral fins along flagellum has been documented in a representative of Characiformes.

Evolutionary trend for metamery reduction and gonad shortening in Anurans revealed by comparison of gonad development.

The gonads develop as the metameric mesodermal structures at the ventral surface of the mesonephroi. To study the evolutionary trends for anuran gonads, we performed comparative analysis of three species from the basal paraphyletic group - Archaeobatrachia (Bombina bombina, Xenopus laevis, Pelobates fuscus) and five species from more derived monophyletic Neobatrachia group (Hyla arborea, Bufotes viridis, Rana dalmatina, Rana arvalis, Rana temporaria). Light and scanning electron microscopy analysis showed that in Archaeobtrachia the undifferentiated gonads and ovaries, but not the testes, have pronounced external metamery. In contrast, Neobatrachia lacked external gonadal metamery and the internal metamery was well pronounced in the ovaries, but only rudimentary in the testes. There was also a difference in the site of genital ridge formation between these two groups. In Archaeobatrachia, the genital ridges developed along almost the entire length of mesonephroi, while in Neobatrachia they were limited to the anterior parts of the mesonephroi. A transient form was observed in P. fuscus, where the gonads formed along the anterior half of the mesonephroi. There was also an evolutionary trend for gradual postero-anterior shortening of the gonadal anlagen. In summary, our study indicates that among anurans there is an evolutionary trend for the gradual loss of metamery and shortening of the gonad.

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.

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.

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 Bidder's organ containing oocytes both in males and females.

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.

Copper metabolism disorders affect testes structure and gamete quality in male mice.

In the present study, animals with a genetic defect in copper metabolism were used as a model organism to study the role of copper in reproduction and to determine whether the disturbances in copper and zinc metabolism affect the testicular tissue and gamete quality in males. Mice with an X-linked mosaic mutation (Atp7a(mo-ms)) exhibit pathological features characteristic of affected copper metabolism. This mutation usually leads to lethality of the mutant males which generally expire on about day 16. Only 4% of mutant animals survive the critical period, achieve maturity, and become fertile. To improve the mutants' viability they were treated with subcutaneous injections of cupric chloride. We measured copper and zinc concentration in the gonads of young (14-day-old) and adult (5-month-old) mutant and control males. Results indicate that copper content was increased but zinc was decreased in the mutant testes. Analysis of the morphology of the testis of the young animals indicate that apoptosis (characteristic for the gonads of young males) was increased in the gonads of the 14-day-old mutants. This process was less advanced in the group of 14-day-old copper treated control males. Apoptosis was also increased in the testes of the adult mutants. Moreover in adult mutants we observed pathological changes in testes morphology (atrophic and sclerotic tubules). Copper and zinc disorders also negatively influenced semen quality parameters, including sperm motility, head morphology, tail cytoplasmic membrane integrity, and number of viable spermatozoa. Poor semen quality of the mutant males seems to be responsible for affected in vivo fertilization efficiency. Treatment with cupric chloride did not influence semen quality except in maturation rate, which was even slower in both mutant and control males after treatment. Additionally, in mutants, copulatory plugs and fertile copulation outcome were decreased after copper treatment.