Testis differentiation in anuran amphibians is the result of two opposing processes: degeneration of the distal part, and development of the proximal part, which becomes a functional male gonad. Undifferentiated gonad differentiates directly into a testis without a transition phase. We described the morphology of developing testes in Rana temporaria and Hyla arborea, and made careful histology and ultrastructure in Pelophylax lessonae. The developing testis was divided into 10 stages (I–III, undifferentiated gonad, IV–X, testis). The earliest morphological symptoms of testis differentiation were observed in 4- to 5-week-old tadpoles at Gosner stage 27–28. At that time an undifferentiated gonad, composed of 6–9 metameres, differentiates into a testis. The proximal metameres (2–3 in the right gonad and 3–4 in the left one) differentiate into a functional testis, while the distal ones degenerate. The difference between left and right gonad size is maintained until adulthood (stage X). Degeneration of the dis...

DNA elimination is a radical form of gene silencing and occurs both in somatic and germ cells. The programmed DNA elimination occurs during gametogenesis in interspecies hybrids that reproduce by hybridogenesis (stick insects, fishes, and amphibians) and concerns removal of whole genomes of one of the parental species and production of clonal gametes propagating the genome of the other species. The cellular mechanisms differ considerably in hybridogenetic insects and fishes but remains unknown in edible frogsPelophylax esculentus, natural hybrids betweenPelophylax lessonaeandPelophylax ridibundus. Here we report DNA elimination mechanism in early developing gonads of diploid and triploid hybrid frogs, studied by TEM, immunofluorescence, and cytochemistry. In gonocytes of both sexes (primary oogonia and prespermatogonia), micronuclei emerge as detached nuclear buds formed during interphase. We found depletion of nuclear pore complexes in micronuclear membrane and chromatin inactivation via heterochromatinization followed by degradation of micronuclei by autophagy. Micronuclei formation does not lead to apoptotic cell death showing that genome elimination is a physiological process. Chromatin elimination via micronuclei inP. esculentusis unique among hybridogenetic animals and contributes to broadening the knowledge about reproductive modes in animals.

Tissue engineering (TE) is a field of regenerative medicine that has been experiencing a special boom in recent years. Among various materials used as components of 3D scaffolds, naturally formed chitinous materials seem to be especially attractive because of their abundance, non-toxic and eco-friendly character. In this study, chitinous skeleton isolated from the marine sponge Aplysina fistularis (phylum: Porifera) was used for the first time as a support for the cultivation of murine fibroblasts (Balb/3T3), human dermal fibroblasts (NHDF), human keratinocyte (HaCaT), and human neuronal (SH-SY5Y) cells. Characterization techniques such as ATR FTIR, TGA, and μCT, clearly indicate that an interconnected macro-porous, thermostable, pure α-chitin scaffold was obtained after alkali–acid treatment of air-dried marine sponge. The biocompatibility of the naturally formed chitin scaffolds was confirmed by cell attachment and proliferation determined by various microscopic methods (e.g., SEM, TEM, digital microscopy) and specific staining. Our observations show that fibroblasts and keratinocytes form clusters on scaffolds that resemble a skin structure, including the occurrence of desmosomes in keratinocyte cells. The results obtained here suggest that the chitinous scaffold from the marine sponge A. fistularis is a promising biomaterial for future research about tissues regeneration.

Glioblastoma is one of the most aggressive tumours with a poor response to treatment and a poor prognosis for patients. One of the proteins expressed in glioblastoma tissue is CHI3L1 (YKL-40), which is upregulated and known for its angiogenesis-supporting and pro-tumour immunomodulatory effects in a variety of cancers. In this paper we present the anti-angiogenic, anti-migratory and immunomodulatory effects of the compound G721-0282, an inhibitor of CHI3L1. The inhibitor-induced changes were investigated using conventional techniques as well as the novel label-free digital holographic tomography (DHT), a quantitative phase imaging technique that allows the reconstruction of the refractive index (RI), which is used as an image contrast for 3D visualisation of living cells. DHT allowed digital staining of individual cells and intercellular structures based only on their specific RI. Quantitative spatially resolved analysis of the RI data shows that the concentration of G721-0282 leads to significant changes in the density of cells and their intracellular structures (in particular the cytoplasm and nucleus), in the volume of lipid droplets and in protein concentrations. Studies in the U-87 MG glioblastoma cell line, THP-1 monocytes differentiated into macrophages, human microvascular endothelial cells (HMEC-1) and in the spheroid model of glioblastoma composed of U-87 MG, HMEC-1 and macrophages suggest that inhibition of CHI3L1 may have potential in the antitumour treatment of glioblastoma. In this paper, we also propose a spheroid model for in vitro studies that mimics this type of tumour.

In our review we have completed current knowledge on myotomal myogenesis in model and non-model vertebrate species (fishes, amphibians, reptiles, birds and mammals) at morphological and molecular levels. Data obtained from these studies reveal distinct similarities and differences between amniote and anamniote species. Based on the available data, we decided to present evolutionary implications in vertebrate trunk muscle development. Despite the fact that in all vertebrates muscle fibres are multinucleated, the pathways leading to them vary between vertebrate taxa. In fishes during early myogenesis myoblasts differentiate into multinucleated lamellae or multinucleate myotubes. In amphibians, myoblasts fuse to form multinucleated myotubes or, bypassing fusion, directly differentiate into mononucleated myotubes. Furthermore, mononucleated myotubes were also observed during primary myogenesis in amniotes. The mononucleated state of myogenic cells could be considered as an old phylogenetic, plesiomorphic feature, whereas direct multinuclearity of myotubes has a synapomorphic character. On the other hand, the explanation of this phenomenon could also be linked to the environmental conditions in which animals develop. The similarities observed in vertebrate myogenesis might result from a conservative myogenic programme governed by the Pax3/Pax7 and myogenic regulatory factor (MRF) network, whereas differences in anamniotes and amniotes are established by spatiotemporal pattern expression of MRFs during muscle differentiation and/or environmental conditions.