Sturgeons are among the most ancient linages of actinopterygians. At present, many sturgeon species are critically endangered. Surrogate production could be used as an affordable and a time-efficient method for endangered sturgeons. Our study established a method for identifying and isolating type A spermatogonia from different developmental stages of testes using flow cytometric cell sorting (FCM). Flow cytometric analysis of a whole testicular cell suspension showed several well-distinguished cell populations formed according to different values of light scatter parameters. FCM of these different cell populations was performed directly on glass slides for further immunocytochemistry to identify germ cells. Results showed that the cell population in gate P1 on a flow cytometry plot (with high forward scatter and high side scatter parameter values) contains the highest amount of type A spermatogonia. The sorted cell populations were characterized by expression profiles of 10 germ cell specific genes. The result confirmed that setting up for the P1 gate could precisely sort type A spermatogonia in all tested testicular developmental stages. The P2 gate, which was with lower forward scatter and side scatter values mostly, contained type B spermatogonia at a later maturing stage. Moreover, expressions of plzf, dnd, boule, and kitr were significantly higher in type A spermatogonia than in later developed germ cells. In addition, plzf was firstly found as a reliable marker to identify type A spermatogonia, which filled the gap of identification of spermatogonial stem cells in sterlet. It is expected to increase the efficiency of germ stem cell culture and transplantation with plzf identification. Our study thus first addressed a phenotypic characterization of a pure type A spermatogonia population in sterlet. FCM strategy can improve the production of sturgeons with surrogate broodstock and further the analysis of the cellular and molecular mechanisms of sturgeon germ cell development.

• Klíčová slova
• PLZF, fluorescence-activated cell sorting, germ stem cell, gonad, spermatogonia, sturgeon,
• Publikační typ
• časopisecké články MeSH

Dead end (dnd) is a germ plasm-specific maternal RNA discovered in zebrafish and then in other vertebrates. Dnd protein is essential for migration and motility of primordial germ cells (PGCs), only cells destined to transfer genetic information to offspring. PGCs arise far from somatic cells of developing gonads and they must migrate to their site of function. Migration of PGCs follows complex path by various developing tissues as their disruption impacts on the fertility. Recently, it has been found that dnd is not required for survival of PGCs and dnd-deficient zebrafish PGCs transdifferentiate into the somatic cells. In fish, targeting dnd causes removal of PGCs that ultimately affects sex differentiation. Sterility in various fish species can be achieved by knockdown or knockout of dnd. In our review, we have discussed dnd as a germ cell-specific molecular marker in fish, its interaction with miRNAs, and its use in aquaculture and fish conservation.

• Klíčová slova
• Aquaculture, Dnd protein, PGCs, Sterilization, Surrogate production,
• MeSH
• lidé MeSH
• mikro RNA MeSH
• pohyb buněk MeSH
• proteiny vázající RNA fyziologie   MeSH
• rybí proteiny fyziologie   MeSH
• ryby MeSH
• vodní hospodářství MeSH
• zárodečné buňky fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• mikro RNA MeSH
• proteiny vázající RNA MeSH
• rybí proteiny MeSH

Genetic introgression of escaped farmed Atlantic salmon (Salmo salar) into wild populations is a major environmental concern for the salmon aquaculture industry. Using sterile fish in commercial aquaculture operations is, therefore, a sustainable strategy for bio-containment. So far, the only commercially used methodology for producing sterile fish is triploidization. However, triploid fish are less robust. A novel approach in which to achieve sterility is to produce germ cell-free salmon, which can be accomplished by knocking out the dead-end (dnd) gene using CRISPR-Cas9. The lack of germ cells in the resulting dnd crispants, thus, prevents reproduction and inhibits subsequent large-scale production of sterile fish. Here, we report a rescue approach for producing germ cells in Atlantic salmon dnd crispants. To achieve this, we co-injected the wild-type (wt) variant of salmon dnd mRNA together with CRISPR-Cas9 constructs targeting dnd into 1-cell stage embryos. We found that rescued one-year-old fish contained germ cells, type A spermatogonia in males and previtellogenic primary oocytes in females. The method presented here opens a possibility for large-scale production of germ-cell free Atlantic salmon offspring through the genetically sterile broodstock which can pass the sterility trait on the next generation.

• MeSH
• CRISPR-Cas systémy MeSH
• genová introgrese genetika   MeSH
• infertilita genetika   MeSH
• kvantitativní znak dědičný MeSH
• oocyty MeSH
• proteiny vázající RNA genetika   MeSH
• rybářství * MeSH
• Salmo salar embryologie   genetika   MeSH
• spermatogonie MeSH
• triploidie MeSH
• zárodečné buňky * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny vázající RNA MeSH

Nanoparticles are finding increasing applications in diagnostics, imaging and therapeutics in medicine. Iron oxide nanoparticles (IONs) have received significant interest of scientific community due to their distinctive properties. For the first time, we have delivered IONs into germ cells in any species. Our results showed that sturgeon primordial germ cells (PGCs) delivered with IONs could be detected until seven days post fertilization (dpf) under fluorescent microscope and at 22 dpf by micro-CT. Delivery of IONs into cells could be helpful for studying germ cell biology and the improvement of germ cell-based bio-technologies as isolation of PGCs using magnetic activated cell sorting or application of hyperthermia for a host sterilization purpose. Intriguingly, in our study, we did not find any toxic effects of IONs on the survival and hatching rates of sturgeon embryos when compared with embryos injected with FITC-dextran only.

• Klíčová slova
• Acipenser, caviar, hyperthermia, iron oxide nanoparticles, micro-CT, sterilization,
• MeSH
• nanočástice * MeSH
• ovum metabolismus   MeSH
• rentgenová mikrotomografie MeSH
• ryby metabolismus   MeSH
• spermie metabolismus   MeSH
• železité sloučeniny chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferric oxide MeSH Prohlížeč
• železité sloučeniny MeSH

Common carp (Cyprinus carpio) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation. However, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me2SO). In the next experiment, a wide range of cooling rates (0.5-10°C/min) and different concentrations of Me2SO were tested resulting in the highest survival achieved using 2 M Me2SO and cooling rate of -1°C/min. When testing different tissue sizes and incubation times in the cryomedia, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the germ cells in gonads of goldfish recipient was confirmed by molecular markers and incorporation rate was over 40% at 3 months post-transplantation. Results of this study can serve for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.

• MeSH
• časové faktory MeSH
• dimethylsulfoxid farmakologie   MeSH
• kapři * MeSH
• kryoprezervace * MeSH
• spermatogonie * cytologie   transplantace   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dimethylsulfoxid MeSH

Sturgeons also known as living fossils are facing threats to their survival due to overfishing and interference in natural habitats. Sterlet (Acipenser ruthenus) due to its rapid reproductive cycle and small body size can be used as a sterile host for surrogate production for late maturing and large sturgeon species. Dead end protein (dnd1) is essential for migration of Primordial Germ Cells (PGCs), the origin of all germ cells in developing embryos. Knockout or knockdown of dnd1 can be done in order to mismigrate PGCs. Previously we have used MO and UV for the aforementioned purpose, and in our present study we have used CRISPR/Cas9 technology to knockout dnd1. No or a smaller number of PGCs were detected in crispants, and we also observed malformations in some CRISPR/Cas9 injected embryos. Furthermore, we compared three established methods to achieve sterility in sterlet, and we found higher embryo survival and hatching rates in CRISPR/Cas9, UV and MO, respectively.

• Klíčová slova
• Acipenser, PGCs, caviar, conservation, genome editing, morpholino oligonucleotide,
• Publikační typ
• časopisecké články MeSH

A technique for rescuing and propagating endangered species involves implanting germ line stem cells into surrogates of a host species whose primordial germ cells (PGCs) have been destroyed. We induced sterilization in sterlet (Acipenser ruthenus) embryos by means of ultraviolet (UV) irradiation at the vegetal pole, the source of early-stage PGCs of sturgeon eggs. The optimal cell stage and length of UV irradiation for the effective repression of the developing PGCs were determined by exposing embryos at the one- to four-cell stage to different doses of irradiation at a wavelength of 254 nm (the optimal absorbance spectrum for germplasm destruction). The vegetal pole region of the embryos was labeled immediately upon irradiation with GFP bucky ball mRNA to monitor the amount of germ plasm and FITC-dextran (M.W. 500,000) to obtain the number of PGCs in the embryos. The size of the germ plasm and number of surrounding mitochondria in the irradiated embryos and controls were observed using transmission electron microscopy, which revealed a drastic reduction in both on the surface of the vegetal pole in the treated embryos. Furthermore, the reduction in the number of PGCs was proportional to the dose of UV irradiation. Under the conditions tested, optimum irradiation for PGCs removal was seen at 360 mJ/cm2 at the one-cell stage. Although some PGCs were observed after the UV irradiation, they significantly reduced in number as the embryos grew. We conclude that UV irradiation is a useful and efficient technique to induce sterility in surrogate sturgeons.

• MeSH
• embryo nesavčí účinky záření   MeSH
• embryonální zárodečné buňky účinky záření   transplantace   MeSH
• ohrožené druhy * MeSH
• ryby embryologie   MeSH
• sterilizace reprodukční metody   veterinární   MeSH
• ultrafialové záření * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Sturgeons (Acipenseriformes) are among the most endangered species in the world due to fragmentation and destruction of their natural habitats and to overexploitation, mainly for highly priced caviar. This has led to the development of sturgeon culture, originally for reintroduction, but more recently for caviar production. In both cases, accurate species identification is essential. We report a new tool for accurate identification of Huso huso and Acipenser ruthenus based on nuclear DNA markers. We employed ddRAD sequencing to identify species-specific nucleotide variants, which served as specific binding sites for diagnostic primers. The primers allowed identification of Huso huso and Acipenser ruthenus as well as their discrimination from A. baerii, A. schrenckii, A. gueldenstaedtii, A. stellatus, A. persicus, A. mikadoi, A. transmontanus, and H. dauricus and identification of A. ruthenus and H. huso hybrids with these species, except hybrid between A. ruthenus and A. stellatus. The species-specific primers also allowed identification of bester (H. huso × A. ruthenus), the most commercially exploited sturgeon hybrid. The tool, based on simple PCR and gel electrophoresis, is rapid, inexpensive, and reproducible. It will contribute to conservation of remaining wild populations of A. ruthenus and H. huso, as well as to traceability of their products.

• MeSH
• buněčné jádro genetika   MeSH
• DNA genetika   MeSH
• druhová specificita MeSH
• genetické markery MeSH
• hybridizace genetická * MeSH
• ryby genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• genetické markery MeSH