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DNA damage during early life stages may have a negative effect on embryo development, inducing malformations that have long-lasting effects during adult life. Therefore, in the current study, we analyzed the effect of DNA damage induced by genotoxicants (camptothecin (CPT) and olaparib) at different stages of embryo development. We analyzed the survival, DNA fragmentation, transcriptome, and proteome of the endangered sturgeon Acipenser ruthenus. Sturgeons are non-model fish species that can provide new insights into the DNA damage response and embryo development. The transcriptomic and proteomic patterns changed significantly after exposure to genotoxicants in a stage-dependent manner. The results of this study indicate a correlation between phenotype formation and changes in transcriptomic and proteomic profiles. CPT and olaparib downregulated oxidative phosphorylation and metabolic pathways, and upregulated pathways involved in nucleotide excision repair, base excision repair, and homologous recombination. We observed the upregulated expression of zona pellucida sperm-binding proteins in all treatment groups, as well as the upregulation of several glycolytic enzymes. The analysis of gene expression revealed several markers of DNA damage response and adaptive stress-response, which could be applied in toxicological studies on fish embryo. This study is the first complex analysis of the DNA damage response in endangered sturgeons.

International audience; In recent years, pollution of surface waters with xenobiotic compounds became an issue of concern in society and has been the object of numerous studies. Most of these xenobiotic compounds are man-made molecules and some of them are qualified as endocrine disrupting chemicals (EDCs) when they interfere with hormones actions. Several studies have investigated the teratogenic impacts of EDCs in vertebrates (including marine vertebrates). However, the impact of such EDCs on marine invertebrates is much debated and still largely obscure. In addition, DNA-altering genotoxicants can induce embryonic malformations. The goal of this study is to develop a reliable and effective test for assessing toxicity of chemicals using embryos of the ascidian (Phallusia mammillata) in order to find phenotypic signatures associated with xenobiotics. We evaluated embryonic malformations with high-content analysis of larval phenotypes by scoring several quantitative and qualitative morphometric endpoints on a single image of Phallusia tadpole larvae with semi-automated image analysis. Using this approach we screened different classes of toxicants including genotoxicants, known or suspected EDCs and nuclear receptors (NRs) ligands. Manuscript File Click here to view linked References 2 The screen presented here reveals a specific phenotypic signature for ligands of retinoic acid receptor/retinoid X receptor. Analysis of larval morphology combined with DNA staining revealed that embryos with DNA aberrations displayed severe malformations affecting multiple aspects of embryonic development. In contrast EDCs exposure induced no or little DNA aberrations and affected mainly neural development. Therefore the ascidian embryo/larval assay presented here can allow to distinguish the type of teratogenicity induced by different classes of toxicants.

DNA damage caused by exogenous or endogenous factors is a common challenge for developing fish embryos. DNA damage repair (DDR) pathways help organisms minimize adverse effects of DNA alterations. In terms of DNA repair mechanisms, sturgeons represent a particularly interesting model due to their exceptional genome plasticity. Sterlet (Acipenser ruthenus) is a relatively small species of sturgeon. The goal of this study was to assess the sensitivity of sterlet embryos to model genotoxicants (camptothecin, etoposide, and benzo[a]pyrene), and to assess DDR responses. We assessed the effects of genotoxicants on embryo survival, hatching rate, DNA fragmentation, gene expression, and phosphorylation of H2AX and ATM kinase. Exposure of sterlet embryos to 1 &micro, M benzo[a]pyrene induced low levels of DNA damage accompanied by ATM phosphorylation and xpc gene expression. Conversely, 20 &micro, M etoposide exposure induced DNA damage without activation of known DDR pathways. Effects of 10 nM camptothecin on embryo development were stage-specific, with early stages, before gastrulation, being most sensitive. Overall, this study provides foundational information for future investigation of sterlet DDR pathways.

In recent years, the development and implementation of new in vitro techniques for ecotoxicology and environmental risk assessment has become a priority; however, the current data indicate a lack of standardization in available in vitro sperm tests. In this review, we describe the main approaches currently used for in vitro toxicity testing of fish spermatozoa. Though fish species show huge diversity in their reproductive strategies, most are external fertilizers. Fish spermatozoa are highly specialized, transcriptionally inactive cells that become motile only upon spawning in the aqueous environment. Here, we suggest that they could become a useful toxicology tool because of their simplicity and availability. Indeed, fish spermatozoa are a promising model for genotoxicity testing, since parental exposure to genotoxicants, both in vivo and in vitro, affect embryo development. In aquaculture practice it means that evaluation of spermatozoa DNA damage could help in predicting embryo quality. Fish spermatozoa could also be useful for the study on nongenomic mechanisms of toxicity, such as the effects of xenobiotics on protein posttranslational modifications. Nevertheless, we conclude that further studies on fish spermatozoa are required to establish a clear correlation between in vitro and in vivo toxicities.

Spermatozoa of externally fertilizing freshwater fish possess several different modes of motility activation. Spermatozoa of common carp (Cyprinus carpioL.) are activated by hypoosmolality, whereas spermatozoa of sterlet (Acipenser ruthenus) require Ca2+ and low concentration of K+ for motility activation. Intracellular signaling differs between these two species as well, particularly in terms of utilization of secondary messengers (cAMP and Ca2+), and kinase activities. The current study was performed in order to determine the importance of protein phosphorylation and protein kinases for activation of sperm motility in carp and sterlet. Treatment with kinase inhibitors indicates that protein kinases A and C (PKA and PKC) participate in spermatozoa motility of both species. Immunodetection of phospho-(Ser/Thr) PKA substrates shows that phosphorylated proteins are localized differently in spermatozoa of carp and sterlet. Strong phosphorylation of PKC substrate was observed in flagella of sterlet spermatozoa, whereas in carp sperm, PKC substrates were lightly phosphorylated in the midpiece and flagella. Motility activation induced either phosphorylation or dephosphorylation on serine, threonine and tyrosine residues of numerous proteins in carp and sterlet spermatozoa. Proteomic methods were used to identify proteins whose phosphorylation state changes upon the initiation of sperm motility. Numerous mitochondrial and glycolytic enzymes were identified in spermatozoa of both species, as well as axonemal proteins, heat shock proteins, septins and calcium-binding proteins. Our results contribute to an understanding of the roles of signaling molecules, protein kinases and protein phosphorylation in motility activation and regulation of two valuable fish species,C. carpioandA. ruthenus.

Main features specific to sperm of fish with external fertilization are 1—the seminal fluid osmolality, and in several species, some ions prevent sperm motility in the genital track; 2—transfer from seminal fluid into sea- or freshwater triggers full motility due to osmotic and/or ionic signal; 3—immediately after activation, fish sperm swim with very high efficiency; 4—the motility period is limited to a very short duration for freshwater sperm and not for much longer in case of marine fish spermatozoa; 5—most motility parameters are decreasing and wave shape is changing during the motility period; 6—the regulation of axonemal motility by ionic concentration, as well as by ATP concentration and other substances, can be observed in vitro by using membrane-deprived spermatozoa; 7—the chemical energy available in fish spermatozoa is rapidly exhausted during the swimming period; 8—the membrane potential of the spermatozoon represents an important aspect of the cell electro-chemical energy homeostasis; 9—the ultimate task for sperm, before fertilization, is to meet an egg, and this task is facilitated by chemotaxis. The present chapter will tentatively deal with some of these salient features such as a—osmolality and ionic signals controlling fast but short-lasting motility in connection with ATP and other energetic compounds management; b—role of sperm electro-chemical potential in the trans -membrane management of this complementary aspect of sperm energetics; c—considerations on physical energy needed by moving fish flagella; and finally, d—how oriented motility governs sperm behavior while swimming toward egg. Finally, recent studies on applied aspects of fish sperm bioenergetics are summarized.

The endocrine disruptor Bisphenol A (BPA), a widely employed molecule in plastics, has been shown to affect several biological processes in vertebrates, mostly via binding to nuclear receptors. Neurodevelopmental effects of BPA have been documented in vertebrates and linked to neurodevelopmental disorders, probably because some nuclear receptors are present in the vertebrate brain. Similarly, endocrine disruptors have been shown to affect neurodevelopment in marine invertebrates such as ascidians, mollusks or echinoderms, but whether invertebrate nuclear receptors are involved in the mode-of-action is largely unknown. In this study, we assessed the effect of BPA on larval brain development of the ascidian Phallusia mammillata. We found that BPA is toxic to P. mammillata embryos in a dose-dependent manner (EC50: 11.8μM; LC50: 21μM). Furthermore, micromolar doses of BPA impaired differentiation of the ascidian pigmented cells, by inhibiting otolith movement within the sensory vesicle. We further show that this phenotype is specific to other two bisphenols (BPE and BPF) over a bisphenyl (2,2 DPP). Because in vertebrates the estrogen-related receptor gamma (ERRγ) can bind bisphenols with high affinity but not bisphenyls, we tested whether the ascidian ERR participates in the neurodevelopmental phenotype induced by BPA. Interestingly, P. mammillata ERR is expressed in the larval brain, adjacent to the differentiating otolith. Furthermore, antagonists of vertebrate ERRs also inhibited the otolith movement but not pigmentation. Together our observations suggest that BPA may affect ascidian otolith differentiation by altering Pm-ERR activity whereas otolith pigmentation defects might be due to the known inhibitory effect of bisphenols on tyrosinase enzymatic activity.

Endocrine Disrupting Chemicals (EDCs) are molecules able to interfere with the vertebrate hormonal system in different ways, a major one being the modification of the activity of nuclear receptors (NRs). Several NRs are expressed in the vertebrate brain during embryonic development and these NRs are suspected to be responsible for the neurodevelopmental defects induced by exposure to EDCs in fishes or amphibians and to participate in several neurodevelopmental disorders observed in humans. Known EDCs exert toxicity not only on vertebrate forms of marine life but also on marine invertebrates. However, because hormonal systems of invertebrates are poorly understood, it is not clear whether the teratogenic effects of known EDCs are because of endocrine disruption. The most conserved actors of endocrine systems are the NRs which are present in all metazoan genomes but their functions in invertebrate organisms are still insufficiently characterized. EDCs like bisphenol A have recently been shown to affect neurodevelopment in marine invertebrate chordates called ascidians. Because such phenotypes can be mediated by NRs expressed in the ascidian embryo, we review all the information available about NRs expression during ascidian embryogenesis and discuss their possible involvement in the neurodevelopmental phenotypes induced by EDCs.

The effect of reactive oxygen species production on the motility parameters, DNA integrity, acid phosphatase activity, and protein tyrosine phosphorylation in spermatozoa of the common carp (Cyprinus carpio) was investigated. Spermatozoa were exposed to different concentrations of xanthine and xanthine oxidase (X-XO) either in the presence or absence of antioxidants for 15 and 60 min. A dose- and time-dependent reduction in spermatozoa motility and velocity was observed. Comet assays showed a dramatic increase in DNA fragmentation after 15 min. Changes in tyrosine phosphorylation of spermatozoa proteins were observed by Western blotting with anti-phosphotyrosine antibodies, and proteins of interest were identified by mass spectrometry. After a 60 min exposure to X-XO, O-linked N-acetylglucosamine transferase, isoform 4 was phosphorylated and septin-8-A was dephosphorylated. Acid phosphatase activity also decreased in a dose-dependent manner after a 60 min exposure to oxidative stress. The results demonstrate that oxidative stress impaired functional variables (sperm motility, velocity, DNA integrity) of carp spermatozoa, and altered intracellular signalling pathways through changes in tyrosine phosphorylation and acid phosphatase activity.

Ascidians (tunicates; sea squirts) are marine animals which provide a source of diverse, bioactive natural products, and a model for toxicity screenings. Compounds isolated from ascidians comprise an approved anti-tumor drug and many others are potent drug leads. Furthermore, the use of invertebrate embryos for toxicological screening tests or analysis offers the possibility to image a large number of samples for high throughput screens. Ascidians are members of a sister clade to the vertebrates and make a vertebrate-like tadpole larva composed of less than 3000 cells in 18 hours. The neural complex of the ascidian larva is made of only 350 cells (of which 100 are neurons) and functional genomic studies have now uncovered numerous GRNs underpinning neural specification and differentiation. Numerous studies showed that brain formation in ascidians is sensitive to toxic insults especially from endocrine disruptors making them a suitable model to study neurodevelopmental defects. Modern techniques available for ascidians, including transgenic embryos where 3D time lapse imaging of GFPexpressing reporter constructs can be analyzed, now permit numerous end-points to be evaluated in order to test the specific mode of action of many compounds. This review summarizes the key evidence suggesting that ascidian embryos are a favorable embryological model to study neurodevelopmental toxicity of different compounds with molecular and cellular end-points. We predict that ascidians may become a significant source of marine blue biotechnologies in the 21st century.

The protective influence of seminal plasma and the antioxidants catalase (CAT), superoxide dismutase (SOD), and glutathione (GTH) on quality parameters, oxidative stress indices, and antioxidant activity was studied in common carp (Cyprinus carpio) spermatozoa exposed to the xanthine-xanthine oxidase (X-XO) system. Fish spermatozoa were incubated for 5 and 20 min at 4 °C with X-XO concentrations of 1 mM X-0.1 U/mL, 0.6 mM X-0.05 U/mL, 0.3 mM X-0.025 U/mL, and 0.1 mM X-0.0125 U/mL. A dose-dependent reduction in spermatozoa motility and velocity was observed at concentrations of 0.1 mM X-0.0125 U/mL to 1 mM X-0.1 U/mL XO. Increase in spermatozoa motility parameters was recorded following treatment with antioxidants and seminal plasma. The level of the oxidative stress indices lipid peroxidation (LPO) and carbonyl derivatives of proteins (CP) was significantly reduced after addition of CAT, SOD, or GTH along with seminal plasma. Significant differences in SOD, glutathione reductase, and glutathione peroxidase activity were seen in spermatozoa incubated with, compared to that without, seminal plasma at all studied X-XO concentrations. The data demonstrated that CAT, SOD, or GTH in combination with SP can reduce reactive oxygen species stress in fish spermatozoa and improve spermatozoa quality.

The sperm of sterlet (Acispenser ruthenus) was used to investigate the effect of the xenobiotic tetrabrombisphenol A (TBBPA) on sperm quality variables (ATP content, spermatozoa motility, and velocity), DNA integrity, and oxidative stress indices. Sperm was diluted to obtain a spermatozoa density of 5 × 108 cells/mL and exposed for 2 h to final concentrations of TBBPA (0.5, 1.75, 2.5, 5, and 10 μg/L). The oxidative stress indices, including lipid peroxidation, carbonyl derivatives of proteins, and antioxidant activity were significantly higher with increased concentrations of TBBPA. There was significantly less intracellular ATP in sperm samples at TBBPA concentrations of 2.5 μg/L and above. Spermatozoa velocity and percent motile sperm were significantly lower at each sampling time post-activation compared to controls. DNA damage expressed as percent DNA in Tail and Olive Tail moment was significantly higher with exposures ≥2.5 μg/L TBBPA. The results demonstrated that TBBPA and other xenobiotics can induce reactive oxygen species stress in fish spermatozoa, which could impair the sperm quality, DNA integrity, ATP content, and the antioxidant defense system. This study confirmed that fish spermatozoa can be used in in vitro assays for monitoring residual pollution in aquatic environments. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 735–745, 2015.

Spermatozoa of externally fertilizing freshwater fish possess several different modes of motility activation. Spermatozoa of common carp (

Oxidant and antioxidant activity in seminal plasma was compared with that in spermatozoa of teleost (common carp Cyprinus carpio and brook trout Salvelinus fontinalis) and chondrostean (Russian stur - geon Acipenser gueldenstaedtii, Siberian sturgeon Acipenser baerii, and sterlet Acipenser ruthenus) fishes. No differences were found between seminal plasma and spermatozoa in the level of thiobarbituric-acid-reactive substance (0.24 ± 0.08 to 0.33 ± 0.04 nmol/mg proteins) in Russian sturgeon, Siberian sturgeon , and sterlet. Carbonyl protein concentration was significantly higher in spermatozoa than in seminal plasma of all studied species. Analyzed antioxidants included superoxide dismutase, glutathione reductase, and glutathione peroxi - dase activity. Significant differences ( P < 0.05) were detected between seminal plasma and spermatozoa in total superoxide dismutase (SOD) and glutathione reductase (GR). Total glutathione peroxidase (GPx) activity was significantly higher in brook trout (12.56 ± 3.23 mU/mg proteins) and Russian sturgeon (11.56 ± 3.12 mU/mg proteins) spermatozoa compared to seminal plasma (6.81 ± 1.56 mU/mg proteins in brook trout and 9.56 ± 3.12 mU/mg proteins in Russian sturgeon). This study provides new data on oxidant and antioxidant balance between spermatozoa and seminal plasma that may be of value in the development of methods for artificial reproduction of teleost and chondrostean species.

The sturgeon is a highly endangered fish mostly due to over-fishing, habitat destruction, and water pollution. Nonylphenol (NP), propranolol (PN), and diethylstilbestrol (DES) are multifunctional xenobiotic compounds used in a variety of commercial and industrial products. The mechanism by which these xenobiotic compounds interfere with fish reproduction is not fully elucidated. This study assessed the effect of NP, PN, and DES on motility parameters, membrane integrity, and oxidative/antioxidant status in sterlet Acispenser ruthenus spermatozoa. Spermatozoa were incubated with several concentrations of target substances for 1h. Motility rate and velocity of spermatozoa decreased in the presence of xenobiotics in a dose-dependent manner compared with controls. A significant decrease in membrane integrity was recorded with exposure to 5μM of NP, 25μM of PN, and 50μM of DES. After 1h exposure at higher tested concentrations NP (5-25μM), PN (25-100μM), and DES (50-200μM), oxidative stress was apparent, as reflected by significantly higher levels of protein and lipid oxidation and significantly greater superoxide dismutase activity. The results demonstrated that NP, PN, and DES can induce reactive oxygen species stress in fish spermatozoa, which could impair sperm quality and the antioxidant defence system and decrease the percentage of intact sperm cells.

Human hepatocellular cells Hep G2 were used to investigate the effects of the intake of contaminated fish on oxidative stress. Uptake of heavy metal contaminated fish was mimicked by incubating the cells with a combination of cadmium chloride (Cd2+) as possible contaminant and a combination of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as important fatty acids (FA) specific for fish. The main aim of this study was to determine the effects of these co-incubations (FA, Cd2+) on lipid and protein oxidation. In addition we also evaluated the antioxidant response of the cells using two different methods (SOD and TAC). Pre-incubation with the chosen FA significantly reduced the oxidative stress caused by incubation with Cd2+. We measured an increased level of carbonyl proteins (CP) in the cells pre-incubated with bovine serum albumin (BSA) and post-incubated with Cd2+.

The effect of reactive oxygen species production on the motility parameters, DNA integrity, acid phosphatase activity, and protein tyrosine phosphorylation in spermatozoa of the common carp (Cyprinus carpio) was investigated. Spermatozoa were exposed to different concentrations of xanthine and xanthine oxidase (X-XO) either in the presence or absence of antioxidants for 15 and 60 min. A dose- and time-dependent reduction in spermatozoa motility and velocity was observed. Comet assays showed a dramatic increase in DNA fragmentation after 15 min. Changes in tyrosine phosphorylation of spermatozoa proteins were observed by Western blotting with anti-phosphotyrosine antibodies, and proteins of interest were identified by mass spectrometry. After a 60 min exposure to X-XO, O-linked N-acetylglucosamine transferase, isoform 4 was phosphorylated and septin-8-A was dephosphorylated. Acid phosphatase activity also decreased in a dose-dependent manner after a 60 min exposure to oxidative stress. The results demonstrate that oxidative stress impaired functional variables (sperm motility, velocity, DNA integrity) of carp spermatozoa, and altered intracellular signalling pathways through changes in tyrosine phosphorylation and acid phosphatase activity.

The sperm of sterlet (Acispenser ruthenus) was used to investigate the effect of the xenobiotic tetrabrombisphenol A (TBBPA) on sperm quality variables (ATP content, spermatozoa motility, and velocity), DNA integrity, and oxidative stress indices. Sperm was diluted to obtain a spermatozoa density of 5 × 10(8) cells/mL and exposed for 2 h to final concentrations of TBBPA (0.5, 1.75, 2.5, 5, and 10 μg/L). The oxidative stress indices, including lipid peroxidation, carbonyl derivatives of proteins, and antioxidant activity were significantly higher with increased concentrations of TBBPA. There was significantly less intracellular ATP in sperm samples at TBBPA concentrations of 2.5 μg/L and above. Spermatozoa velocity and percent motile sperm were significantly lower at each sampling time post-activation compared to controls. DNA damage expressed as percent DNA in Tail and Olive Tail moment was significantly higher with exposures ≥2.5 μg/L TBBPA. The results demonstrated that TBBPA and other xenobiotics can induce reactive oxygen species stress in fish spermatozoa, which could impair the sperm quality, DNA integrity, ATP content, and the antioxidant defense system. This study confirmed that fish spermatozoa can be used in in vitro assays for monitoring residual pollution in aquatic environments.

The aim of the study was to examine sperm maturation in sturgeon and to establish the localization of the maturation. We demonstrated that sperm maturation occurs in sturgeon outside the testes via dilution of sperm by urine. The process involves the participation of high molecular weight (>10kDa) substances and calcium ions.

Among endocrine disruptors, the xenoestrogen bisphenol A (BPA) deserves particular attention due to widespread human exposure. Besides hormonal effects, BPA has been suspected to be responsible for adverse effect on reproductive ability of various species. In the present study the effect of BPA on the quality parameters, oxidative stress, the DNA integrity and intracellular ATP content of sterlet (Acipenser ruthenus) spermatozoa were investigated in vitro. Fish spermatozoa were exposed to concentrations of BPA possibly occurring in nature (0.5, 1.75, 2.5, 5 and 10μg/L) for 2h. Results revealed that BPA significantly decreased spermatozoa motility and velocity of spermatozoa at concentration of BPA 2.5-10μg/L. Significant positive correlation (r=0.713, P

The sturgeon is a highly endangered fish species mostly due to over-fishing, habitat destruction, and water pollution. Duroquinone (derivative of 1,4-benzoquinone) is a xenobiotic compound widespread in the environment. The effect of duroquinone on motility, DNA integrity, and oxidative stress indices in sterlet, Acispenser ruthenus, spermatozoa was investigated. Sterlet sperm was exposed for 2h to duroquinone at concentrations of 25, 50, 100, and 150 μM. Spermatozoa motility, velocity, and ATP content were significantly decreased with exposure to duroquinone. The level of DNA damage significantly increased at concentrations of 50 μM and above. Oxidative stress indices (lipid peroxidation and content of carbonyl proteins) and superoxide dismutase (SOD) activity increased significantly with increasing concentrations of duroquinone. Oxidative stress in sterlet spermatozoa induced by duroquinone was shown to impair spermatozoa DNA integrity, motility parameters, and the antioxidant defense system. Spermatozoa motility, content of carbonyl proteins, and SOD activity were shown to be sensitive biomarkers, exhibiting strong responses to low concentrations of the xenobiotic. Results also suggested that fish spermatozoa in vitro assays may provide a simple and efficient means of monitoring residual pollutants in the aquatic environment.

We report, for the first time, a series of baseline techniques comprising isolation and transplantation of female and male early-stage germ cells in sturgeon to generate a germline chimera as a potential tool for surrogate reproduction and gene banking. Cells were dissociated from testis, characterized by mostly spermatogonia, and from ovary, exclusively comprising oogonia and previtellogenic oocytes, of Acipenser baerii, using 0.3% trypsin (2 hours, 23 °C) dissolved in PBS, isotonic with blood plasma. The dissociated germ cells were sorted by Percoll gradient centrifugation followed by immunolabeling with germ cell–specific vasa antibody DDX4, while 10% to 30% Percoll solution contained 79.4% and 70.8% labeled testicular and ovarian cells. Sorted germ cells were transplanted into a cavity close to a presumptive genital ridge of newly hatched heterospecific Acipenser ruthenus larvae with fluorescein isothiocyanate–labeled endogenous primordial germ cells. The transplanted germ cells were randomly distributed in the body cavity through 30-day posttransplantation (dpt). Subsequently, the cells were organized into genital ridges 50 dpt and proliferated 90 dpt. The number of both transplanted and endogenous germ cells significantly increased from 18.1, 22.2, and 29.1 (30 dpt) to 108.5, 90.8, and 118.5 (90 dpt) in ovarian, testicular, and endogenous germ cells, respectively (P

A practical technique for isolation and cryopreservation of tench (Tinca tinca) (Cyprinidae, Teleostei) early stages of germ cells (GC), including spermatogonia and spermatocytes, is reported for the first time. The germ-line cells possess the ability to differentiate into functional gametes of both sexes. These early stages of germ cells are small enough to be well-suited to cryopreservation, which, together with their high level of plasticity, makes their preservation a promising tool for maintaining genetic resources. Testicular cells were distinguished and separated by Percoll gradient, with the highest proportion of GC (62.2%) obtained from the 30% layer. The concentration and viability of GC were determined, and specific staining (DDX4) for germ cells was used to distinguish GC from somatic cells. Early stages of germ cells were cryopreserved in an extender composed of phosphate buffered saline (pH 8) with 0.5% BSA, 50mM d -glucose, and containing 1.5M cryo- protectant in the pre-programmed PLANER Kryo10 series III using a cooling protocol from +10°C to -80°C at a rate of 1°C/min. The effect of six cryoprotectants - methanol, dimethyl sulfoxide, dimethyl sulfoxide + propanediol (1 : 1), glycerol, ethylene glycol, and dimethylacetamid was assessed, and the results were evaluated by comparing the percentage of viable frozen/thawed GC by ANOVA, Tukey's HSD test (P < 0.05). Almost the same viability rates were obtained with no significant differences among tested cryoprotectants, indicating high stability of GC in cryoprotectants. Nevertheless, glycerol at a concentration of 1.5M was associated with the highest survival rate of thawed tench GC (57.69 ± 16.85%).