Identification of Novel Proteins in Foam Nests of the Japanese Forest Green Tree Frog, Rhacophorus arboreus.

Foam nests of frogs are natural biosurfactants that contain potential compounds for biocompatible materials, Drug Delivery System (DDS), emulsifiers, and bioremediation. To elucidate the protein components in the foam nests of Rhacophorus arboreus, which is an endemic Japanese frog species commonly seen during the rainy season, we performed amino acid analysis, SDS-PAGE electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry using intact foam nests. Many proteins were detected in these foam nests, ranging from a few to several hundred kDa, with both essential and non-essential amino acids. Next, we performed transcriptome analysis using a next-generation sequencer on total RNAs extracted from oviducts before egg-laying. The soluble foam nests were purified by LC-MS and analyzed using Edman degradation, and the identified N-terminal sequences were matched to the transcriptome data. Four proteins that shared significant sequence homologies with extracellular superoxide dismutase of Nanorana parkeri, vitelline membrane outer layer protein 1 homolog of Xenopus tropicalis, ranasmurfin of Polypedates leucomystax, and alpha-1-antichymotrypsin of Sorex araneus were identified. Prior to purification of the foam nests, they were treated with both a reducing reagent and an alkylating agent, and LC-MS/ MS analyses were performed. We identified 22 proteins in the foam nests that were homologous with proteinase inhibitors, ribonuclease, glycoproteins, antimicrobial protein and barrier, immunoglobulin-binding proteins, glycoprotein binding protein, colored protein, and keratin-associated protein. The presence of these proteins in foam nests, along with small molecules, such as carbohydrates and sugars, would protect them against microbial and parasitic attack, oxidative stress, and a shortage of moisture.

Complete mitochondrial genome sequence of Japanese forest green tree frog (Rhacophorus arboreus).

We determined the complete mitochondrial genome sequence of the Japanese forest green tree frog (Rhacophorus arboreus). The mitochondrial genome is 22,236 bp in length, which encodes 13 protein-coding genes, 2 rRNA, and 22 tRNA genes, and two control regions (D-loops). The whole gene arrangement of R. arboreus was the same as that of Rhacophorus omeimontis and Rhacophorus schlegelii.

Ultrastructure and motility of the spermatozoa of Polypedates leucomystax (Amphibia, Anura, Rhacophoridae).

Sperm morphology is thought to be shaped by evolutionary pressure from the fertilization environment. Rhacophoridae (Amphibia, Anura) include both foam-nesting and nonfoam-nesting species and exhibit a variety of sperm morphologies. Here, we examine the sperm morphology and motility of a foam-nesting Rhacophoridae frog, Polypedates leucomystax. Their spermatozoa have a sickle-shaped head and a thick tail containing two axonemes with their doublet microtubule ones (Db1s) facing one another. These two axonemes are surrounded by hundreds of satellite microtubules that form a hexagonal lattice structure. The spermatozoa move spirally by directly converting their tail movements into propulsion force, similar to the movement of the sickle-shaped spermatozoa of Xenopus laevis. By comparing the spermatozoa of P. leucomystax to those of other foam-nesting Rhacophoridae frogs, Rhacophorus and Chiromantis, and to the nonfoam-nesting Rhacophoridae frog, Buergeria buergeri, we found the following: (i) Spermatozoa of foam-nesting Rhacophoridae share common morphological features, a pair of axonemes and crystallized satellite microtubules. (ii) Spermatozoa of nonfoam-nesting Rhacophoridae do not exhibit these features. (iii) Sperm motility in foam-nesting Rhacophoridae is adapted to viscous environments. (iv) A diversity of sperm morphology and motility exists even among foam-nesting Rhacophoridae frogs. (v) The spermatozoa of Rhacophorus are more adapted to the foam nest than the spermatozoa of Polypedates.

Multiple functions of FADD in apoptosis, NF-κB-related signaling, and heart development in Xenopus embryos.

FADD is an adaptor protein that transmits apoptotic signals from death receptors. Additionally, FADD has been shown to play a role in various functions including cell proliferation. However, the physiological role of FADD during embryonic development remains to be delineated. Here, we show the novel roles FADD plays in development and the molecular mechanisms of these roles in Xenopus embryos. By whole-mount in situ hybridization and RT-PCR analysis, we observed that fadd is constantly expressed in early embryos. The upregulation or downregulation of FADD proteins by embryonic manipulation resulted in induction of apoptosis or size changes in the heart during development. Expression of a truncated form of FADD, FADDdd, which lacks pro-apoptotic activity, caused growth retardation of embryos associated with dramatic expressional fluctuations of genes that are regulated by NF-κB. Moreover, we isolated a homolog of mammalian cullin-4 (Cul4), a component of the ubiquitin E3 ligase family, as a FADDdd-interacting molecule in Xenopus embryos. Thus, our study shows that FADD has multiple functions in embryos; it plays a part in the regulation of NF-κB activation and heart formation, in addition to apoptosis. Furthermore, our findings provide new insights into how Cul4-based ligase is related to FADD signaling in embryogenesis.

Ultrastructural analysis of spermiogenesis in Rhacophorus arboreus (Amphibia, Anura, Rhacophoridae).

The spermatozoa of the Japanese green tree frog, Rhacophorus arboreus (Amphibia, Anura, Rhacophoridae), have a characteristic corkscrew-shaped head and a thick tail that extends perpendicular to the longitudinal axis of the head. We examined the process of spermatogenesis in Rh. arboreus, particularly spermiogenesis, using light and transmission electron microscopy. Spermiogenesis was categorized into the early, mid, and late stages based on the spermatid morphology and their location within the cyst. Early spermatids had a round nucleus and two independent flagella that elongated from a pair of parallel centrioles. The centrioles became embedded in centriolar adjunct material and attached to the nucleus. Then, the flagella were covered with a mantle-like cytoplasm that contained many microtubules. An acrosome appeared on the pointed side of the slightly elongated nucleus. Mid spermatids had an elongated rod-like head. As the nucleus elongated, the chromatin fibers became thicker and were arranged parallel to the elongation axis. An elongated acrosome was attached helically along the lateral side of the elongated nucleus. The biflagellate spermatids transformed into monoflagellate spermatids with two axonemes through a process in which the plasma membrane of each flagellum expanded. Late spermatids had a coiled or corkscrew-shaped head. An acrosome was located on the inside of the coiled cone composed of a nucleus. Parallel microtubules were connected in rows, and then became crystallized in the tail. The present report contains the first morphological description of spermatogenesis in Rhacophorus and suggests that spermiogenesis evolved to adapt to the fertilization environment.

A novel mechanism of sperm motility in a viscous environment: corkscrew-shaped spermatozoa cruise by spinning.

Fertilization of the green tree frog, Rhacophorus arboreus, occurs in the viscous environment of a foam nest, which is laid on vegetation. Their spermatozoa have a characteristic corkscrew-shaped head and a thick tail that extends perpendicularly to its longitudinal axis. However, it is unclear how these corkscrew-shaped spermatozoa move in this highly viscous environment. Here, we found that the spinning of the corkscrew-shaped head, caused by winding and unwinding of the tail, enables the spermatozoa to move through the highly viscous environment of a foam nest, like a corkscrew rotating into a cork. We suggested that dislocations observed in the matrix of satellite microtubules surrounding two axonemes, reflected the planes of sliding of the axonemes, and dyneins on doublets two and six of each axoneme were active during winding and unwinding, respectively. These results provide a novel mechanism for sperm movement that is adapted specifically to a viscous fertilization environment.

The adaptor molecule FADD from Xenopus laevis demonstrates evolutionary conservation of its pro-apoptotic activity.

FADD is an adaptor protein that transmits apoptotic signals from death receptors such as Fas to downstream initiator caspases in mammals. We have identified and characterized the Xenopus orthologue of mammalian FADD (xFADD). xFADD contains both a death effector domain (DED) and a death domain (DD) that are structurally homologous to those of mammalian FADD. We observed xFADD binding to Xenopus caspase-8 and caspase-10 as well as to human caspase-8 and Fas through interactions with their homophilic DED and DD domains. When over-expressed, xFADD was also able to induce apoptosis in wild-type mouse embryonic fibroblasts (MEF), but not in caspase-8-deficient MEF cells. In contrast, DED-deficient xFADD (xFADDdn) acted as a dominant-negative mutant and prevented Fas-mediated apoptosis in mammalian cell lines. These results indicate that xFADD transmits apoptotic signals from Fas to caspase-8. Furthermore, we found that transgenic animals expressing xFADD in the developing heart or eye under the control of tissue-specific promoters show abnormal phenotypes. Taken together, these results suggest that xFADD can substitute functionally for its mammalian homologue in death receptor-mediated apoptosis, and we suggest that xFADD functions as a pro-apoptotic adaptor molecule in frogs. Thus, the structural and functional similarities between xFADD and mammalian FADD provide evidence that the apoptotic pathways are evolutionally conserved across vertebrate species.

Homoiogenetic Neural Induction in Xenopus Chimeric Explants: (Xenopus laevis/Xenopus borealis/chimeric explants/homoiogenetic neural induction/tissue-specific monoclonal antibodies).

We previously raised monoclonal antibodies specific for epidermis (7) and neural tissue (8) of Xenopus for use as markers of tissue differentiation in induction experiments (8). Here we have used these monoclonal antibodies to examine homoiogenetic neural induction, by which cells induced to differentiate to neural tissues can in turn induce competent ectoderm to do the same. Presumptive anterior neural plate excised from late gastrulae of Xenopus laevis was conjugated with competent ectoderm from the initial gastrula of Xenopus borealis, either side by side or with their inner surfaces together. The chimeric explants enabled us to distinguish induced neural tissues from inducing neural tissues. In both types of explant, neural tissues identified by the neural tissue-specific antibody, NEU-1, were induced in the competent ectoderm by the presumptive anterior neural plate. The results suggest that homoiogenetic neural induction does occur in Xenopus embryos.

Expression of Neural Antigens in Normal Xenopus Embryos and Induced Explants: (Xenopus laevis/neural antigens/monoclonal antibodies/neural induction/neural differentiation).

Monoclonal antibodies were raised against neural tissues of Xenopus larvae. Three monoclonal antibodies, named NEU-1, NEU-3, and NEU-4, were specific for neural tissue and first bound to neural cells at stage 25 after neural tube formation (NEU-1 and NEU-3) or at stage 31 (NEU-4). These antibodies bound to differentiating neural cells, but not to germinal neuroepithelial cells. NEU-1 and NEU-3 recognized antigens in cell bodies as well as neural fibers of neural cells, and these antigens were distributed throughout the central nervous system. NEU-4 bound to antigens in granular materials in neural cells, and these antigens were present in head and trunk regions but not in the tail region. These three antibodies were used as neural markers in two types of induction experiments, in which 1) the animal pole region and the dorsal blastopore lip from stage-10 gastrulae were combined, or 2) the animal pole region and the vegetal pole region from stage-8 blastulae were combined. In both experiments, most conjugated explants expressed the NEU-1, NEU-3, and NEU-4 antigens, although the expression of NEU-4 antigen was delayed compared with those of the NEU-1 and NEU-3 antigens. These results show that these antibodies are useful as markers in neural induction experiments.

Developmental Fates of Blastomeres of Eight-Cell-Stage Xenopus laevis Embryos: (intracellular injection/horseradish peroxidase/developmental fate/Xenopus embryo).

The developmental fates of animal, vegetal, dorsal, and ventral egg regions of Xenopus laevis embryos were examined. For this purpose, a tracer enzyme (horseradish peroxidase) was injected bilaterally into pairs of eight-cell-stage blastomeres and the clonal organization of marked cells in the early tail-bud embryos was examined. The epidermis over most of the body originated from animal-ventral micromeres, but that in the head originated from animal-dorsal blastomeres and that in the area surrounding the anus originated from vegetal-ventral blastomeres. The neural tube originated from animal-dorsal, vegetal-dorsal, and animal-ventral blastomeres. These results were consistent with those of previous studies. But in contrast to previous findings, results showed that the entire notochord is derived from animal-dorsal micromeres and that the somites originate from all four bilateral pairs of blastomeres in the eight-cell stage. These results are discussed in relation to the current maps of prospective fates based on results of vital-dye staining. Morphogenetic movements are also discussed on the basis of the clonal organization demonstrated in the present study.

CYCLIC SURFACE CHANGES IN THE NON-NUCLEATE EGG FRAGMENT OF XENOPUS LAEVIS.

Fertilized uncleaved eggs of Xenopus laevis were divided into nucleate and non-nucleate egg fragments. Both fragments, together with the whole egg of the same batch, were observed by time-lapse cinematography. Two kinds of cyclic surface changes, (1) rounding-up and relaxing movements and (2) surface contraction waves, accompanying each cleavage in the whole eggs and the nucleate fragments, were also observed even in the non-nucleate fragments although they do not cleave. Cleavage intervals of the whole egg and the nucleate fragment were nearly equal, but the rounding-up intervals of the non-nucleate fragment were slightly but definitely longer than the cleavage intervals of the nucleate fragment and the whole egg.