GCN2-eIF2α signaling pathway negatively regulates the growth of triploid crucian carp.

GCN2-eIF2α signaling pathway plays crucial roles in cell growth,development, and protein synthesis. However, in polyploid fish, the function of this pathway is rarely understood. In this study, genes associated with the GCN2-eIF2α pathway (pkr, pek, gcn2, eif2α) are founded lower expression levels in the triploid crucian carp (3nCC) muscle compared to that of the red crucian carp (RCC). In muscle effect stage embryos of the 3nCC, the mRNA levels of this pathway genes are generally lower than those of RCC, excluding hri and fgf21. Inhibiting gcn2 in 3nCC embryos downregulates downstream gene expression (eif2α, atf4, fgf21), accelerating embryonic development. In contrast, overexpressing of eif2α can alter the expression levels of downstream genes (atf4 and fgf21), and decelerates the embryonic development. These results demonstrate the GCN2-eIF2α pathway's regulatory impact on 3nCC growth, advancing understanding of fish rapid growth genetics and offering useful molecular markers for breeding of excellent strains.

Discovery of a novel miRNA involved in the regulation of male infertility in zebrafish.

Azoospermia and asthenospermia are common manifestations of male infertility, but it needs further studies to understand the intrinsic regulation mechanism. As a popular model organism, zebrafish is often used to assess reproductive complications. In this study, by analyzing miRNA transcriptome of the mature triploid zebrafish testis afflicted with spermatogenic dysfunctions, leading to the identification of 36 miRNAs that are differentially expressed in comparison with diploid, which are predicted to target 2737 genes. Subsequent functional annotation of these genes pinpointed two miRNAs might association with spermatogenesis. Inhibitory experiments showed that NC_007115.7.7_998413 inhibited conducts a substantial decline in sperm density, and conducted lower embryo fertilization rate than control. And putative target genes qRT-PCR evaluation showed that spata2 was significant down-regulate upon inhibited NC_007115.7.7_998413. In summary, this research positions newly identified miRNA NC_007115.7.998413 as a regulatory factor in male zebrafish reproductive development, enhancing our comprehension of the molecular regulated pathways involved in spermatogenesis.

Defining the Pluripotent Marker Genes for Identification of Teleost Fish Cell Pluripotency During Reprogramming.

Pluripotency is a transient state in early embryos, which is regulated by an interconnected network of pluripotency-related genes. The pluripotent state itself seems to be highly dynamic, which leads to significant differences in the description of induced pluripotent stem cells from different species at the molecular level. With the application of cell reprogramming technology in fish, the establishment of a set of molecular standards for defining pluripotency will be important for the research and potential application of induced pluripotent stem cells in fish. In this study, by BLAST search and expression pattern analysis, we screen out four pluripotent genes (Oct4, Nanog, Tdgf1, and Gdf3) in zebrafish (Danio rerio) and crucian carp (Carassius). These genes were highly expressed in the short period of early embryonic development, but significantly down-regulated after differentiation. Moreover, three genes (Oct4, Nanog and Tdgf1) have been verified that are suitable for identifying the pluripotency of induced pluripotent stem cells in zebrafish and crucian carp. Our study expands the understanding of the pluripotent markers of induced pluripotent stem cells in fish.

Characteristics of SP600125 Induced Tetraploid Cells in Comparison With Diploid and Tetraploid Cells of Fish.

In our previous research, SP600125 (Anthrapyrazolone) was used to induce autotetraploid of crucian carp cells (SP4N cells), and tetraploid fry was generated from the SP4N cells by somatic cell nuclear transfer technique. However, it is still unclear about biological characteristics of the SP4N cells. In this article, the cytological characteristic and gene expression profiles of the SP4N cells are investigated in comparison with the crucian carp cells (2N cells) and the tetraploid crucian carp cells (CC4N cells). The SP4N cells have tetraploid characteristics in terms of morphology and DNA ploidy levels, and their chromosome behavior is stable during the cell proliferation. The migration ability and the mtDNA copy number of SP4N cells are both lower than those in the CC4N cells and the 2N cells, but there exist giant mitochondria in the SP4N cells. The similar expression trends in the cell cycle regulation genes of the SP4N cells and 2N cells, while the corresponding expression profiles are clearly different between the SP4N cells and the CC4N cells. Moreover, the significant difference genes are associated with energy metabolism pathways among the SP4N cells, 2N cells and CC4N cells. These results can provide deeper understanding of SP600125 induction, as well as finding applications in polyploidization breeding of fish species.

Enhanced Immune Response Improves Resistance to Cadmium Stress in Triploid Crucian Carp.

Previous research has indicated that triploid crucian carp (3n fish) have preferential resistance to cadmium (Cd) compared to Carassius auratas red var. (2n fish). In this article, comparative research is further conducted between the 2n and 3n fish in terms of the immune response to Cd-induced stress. Exposure to 9 mg/L Cd for 96 h changed the hepatic function indexes remarkably in the 2n fish, but not in the 3n fish. In the serum of Cd-treated 2n fish, the levels of alanine amino transferase, aspartate aminotransferase, adenosine deaminase, and total bilirubin significantly increased, while the levels of total protein, albumin, lysozyme, and anti-superoxide anion radicals decreased demonstrating hepatotoxicity. By analysis of transcriptome profiles, many immune-related pathways were found to be involved in the response of 3n fish to the Cd-induced stress. Expression levels of the immune genes, including the interleukin genes, tumor necrosis factor super family member genes, chemokine gene, toll-like receptor gene, and inflammatory marker cyclooxygenase 2 gene were significantly enhanced in the hepatopancreas of the Cd-treated 3n fish. In contrast, the expression levels of these genes decreased in the 2n fish. This research provides a theoretical basis for polyploid fish breeding and is helpful for the ecological restoration of water due to pollution.

Fish Pluripotent Stem-Like Cell Line Induced by Small-Molecule Compounds From Caudal Fin and its Developmental Potentiality.

The technique of induced pluripotent stem cells has significant application value in breeding and preserving the genetic integrity of fish species. However, it is still unclear whether the chemically induced pluripotent stem cells can be induced from non-mammalian cells or not. In this article, we first verify that fibroblasts of fish can be chemically reprogrammed into pluripotent stem cells. These induced pluripotent stem-like cells possess features of colony morphology, expression of pluripotent marker genes, formation of embryoid bodies, teratoma formation, and the potential to differentiate into germ cell-like cells in vitro. Our findings will offer a new way to generate induced pluripotent stem cells in teleost fish and a unique opportunity to breed commercial fish and even save endangered fish species.

Optimal reference genes for gene expression analysis in polyploid of Cyprinus carpio and Carassius auratus.

BACKGROUND: Reference genes are usually stably expressed in various cells and tissues. However, it was reported that the expression of some reference genes may be distinct in different species. In this study, we intend to answer whether the expression of reported traditional reference genes changes or not in the polyploid fish RESULTS: By retrieving the mRNA sequencing data of three different ploidy fish from the NCBI SRA database, we selected 12 candidate reference genes, and examined their expression levels in the 10 tissues and in the four cell lines of three different ploidy fish by real-time PCR. Then, the expression profiles of these 12 candidate reference genes were systematically evaluated by using the software platforms: BestKeeper, NormFinder and geNorm. CONCLUSION: The 28S ribosomal protein S5 gene (RPS5) and the ribosomal protein S18 gene (RPS18) are the most suitable reference genes for the polyploid of Cyprinus carpio and Carassius auratus, demonstrated by both of the tissues and the cultured cells.

Expression Profile Analysis of the Cell Cycle in Diploid and Tetraploid Carassius auratus red var.

Polyploidization often leads to "transcriptome shock," and is considered an important factor in evolution of species. Analysis of the cell cycle, which is associated with survival in polyploidy, has proved useful in investigating polyploidization. Here, we used mRNA sequencing to investigate global expression in vitro (in cultured cells) and in vivo (in fin and liver tissues) in both the diploid and tetraploid Carassius auratus red var.. Differential expression (DE) of genes in diploid (7482, 36.0%) and tetraploid (3787, 18.2%) states suggested that in vitro and in vivo conditions dramatically change mRNA expression levels. However, of the 20,771 total shared expressed genes, 18,050 (87.0%), including 17,905 (86.2%) non-differentially expressed genes (DEGs) and 145 (0.7%) DEGs between diploids and tetraploids, showed the same expression trends in both cultured cells and liver tissues. Of the DEGs, four of seven genes in the cell cycle pathway had the same expression trends (upregulated in diploids and tetraploids) in both cultured cells and liver tissues. Quantitative PCR analysis confirmed the same expression trends in the nine DEGs associated with regulation of the cell cycle. This research on common characteristics between diploids and tetraploids provides insights into the potential molecular regulatory mechanisms of polyploidization. The steady changes that occur between diploids and tetraploids in vitro and in vivo show the potential value of studying polyploidy processes using cultured cell lines, especially with respect to cell cycle regulation.

Bisexual Fertile Triploid Zebrafish (Danio rerio): a Rare Case.

Previous studies have suggested that artificially induced triploid zebrafish are exclusively male-biased. Owing to greatly inhibited gonadal development for the artificially induced triploid fish, they are regarded to be sterile in general. In this article, partially fertile bisexual triploid zebrafish are produced by suppressing extrusion of the second polar body by heat shock. Histological observation confirms that the early gonadal development of these triploid zebrafish is normal. Backcrossing and self-crossing are used to demonstrate that both the female and male triploid zebrafish have partial reproductive ability. Their dynamic of chromosomes during meiosis is revealed from the chromosome preparations of gonads. Examination of the expressed gonadal development-related genes shows some molecular evidence of the normal gonadal development in the triploid zebrafish. Clearly, these fertile bisexual triploid zebrafish can provide a unique system to study sex determination, as well as aneuploidy associated human diseases such as infertility and pregnancy loss.

A New Method for Chromosomes Preparation by ATP-Competitive Inhibitor SP600125 via Enhancement of Endomitosis in Fish.

Previous studies have suggested that 1,9-Pyrazoloanthrone, known as SP600125, can induce cell polyploidization. However, what is the phase of cell cycle arrest caused by SP600125 and the underlying regulation is still an interesting issue to be further addressed. Research in this article shows that SP600125 can block cell cycle progression at the prometaphase of mitosis and cause endomitosis. It is suggested that enhancement of the p53 signaling pathway and weakening of the spindle assembly checkpoint are associated with the SP600125-induced cell cycle arrest. Using preliminary SP600125 treatment, the samples of the cultured fish cells and the fish tissues display a great number of chromosome splitting phases. Summarily, SP600125 can provide a new protocol of chromosomes preparation for karyotype analysis owing to its interference with prometaphase of mitosis.

Optimization Techniques to Deeply Mine the Transcriptomic Profile of the Sub-Genomes in Hybrid Fish Lineage.

It has been shown that reciprocal cross allodiploid lineage with sub-genomes derived from the cross of Megalobrama amblycephala (BSB) × Culter alburnus (TC) generates the variations in phenotypes and genotypes, but it is still a challenge to deeply mine biological information in the transcriptomic profile of this lineage owing to its genomic complexity and lack of efficient data mining methods. In this paper, we establish an optimization model by non-negative matrix factorization approach for deeply mining the transcriptomic profile of the sub-genomes in hybrid fish lineage. A new so-called spectral conjugate gradient algorithm is developed to solve a sequence of large-scale subproblems such that the original complicated model can be efficiently solved. It is shown that the proposed method can provide a satisfactory result of taxonomy for the hybrid fish lineage such that their genetic characteristics are revealed, even for the samples with larger detection errors. Particularly, highly expressed shared genes are found for each class of the fish. The hybrid progeny of TC and BSB displays significant hybrid characteristics. The third generation of TC-BSB hybrid progeny ( B T F 3 and T B F 3 ) shows larger trait separation.

Generation of Stable Induced Pluripotent Stem-like Cells from Adult Zebra Fish Fibroblasts.

Induced pluripotent stem (iPS) cells provide a powerful platform for the study of development, regeneration, and disease. Although many stable iPS cell lines have been established for mammals, few attempts have been made to induce iPS cells in nonmammalian species. Because of technical advantages over other vertebrates on stem cells, induced pluripotent stem cells from fish could be of value for research. In this paper, stable iPS-like cell lines were generated from adult zebra fish fibroblasts by combining the doxycycline inducible lentiviral delivery system and chemical treatment. RT-PCR analysis, alkaline phosphatase staining, and immunofluorescence indicated that adult zebra fish fibroblasts were successfully reprogrammed into iPS-like cells (ziPSCs). The ziPSCs exhibited stable growth and manifested many features of fish embryonic stem cells with pluripotency in vitro and in vivo. Because of easy maintenance, the developed technology in this study for generating zebra fish iPS-like cells could be extended to investigating other genera of fish.

Acute immune stress improves cell resistance to chemical poison damage in SP600125-induced polyploidy of fish cells in vitro.

Previous research has indicated that the small compound, SP600125, could induce polyploidy of fish cells, and has established a stable tetraploid cell line from diploid fish cells. In order to explore how fish cells maintain homeostasis under SP600125-stress in vitro, this study investigates impacts of SP600125-stress on intracellular pathways, as well as on regulation of the cellular homeostasis feedback in fish cells. Transcriptomes are obtained from the SP600125-treated cells. Compared with unigenes expressed in control group (crucial carp fin cells), a total of 2670 and 1846 unigenes are significantly upregulated and downregulated in these cells, respectively. Differentially expressed genes are found, which are involved in innate defense, inflammatory pathways and cell adhesion molecules-related pathways. The SP600125-stress enhances cell-mediated immunity, characterized by significantly increasing expression of multiple immune genes. These enhanced immune genes include the pro-inflammatory cytokines (IL-1ß, TNF-ɑ, IL-6R), the adaptor signal transducers (STAT, IκBɑ), and the integrins (ɑ2ß1, ɑMß2). Furthermore, mitochondria are contributed to the cellular homeostasis regulation upon the SP600125-stress. The results show that acute inflammation is an adaptive and controlled response to the SP600125-stress, which is beneficial for alleviating toxicity by SP600125. They provide a potential way of breeding fish polyploidy induced by SP600125 in the future research.

Enhanced Resistance of Triploid Crucian Carp to Cadmiuminduced Oxidative and Endoplasmic Reticulum Stresses.

BACKGROUND: Cadmium is a major heavy metal pollutant. Even at low concentrations in waste water Cadmium can accumulate in algae and sediments, and it is absorbed by both plants and aquatic animals, like shellfish and fish. Triploid crucian carp is an important economic fish, and have been farmed on a large scale in China. OBJECTIVE: to explore the molecular mechanisms underlying Cadmium stress of triploid crucian carp. METHOD: we applied following experimental method to conduct research: transcriptomes analysis, histological observation, Quantitative real-time PCR and enzyme activity analysis et al. Results: in the present study, we obtained following important results: 1) Under Cd stress, the mortality and abnormality rates in triploid crucian carp were lower than those of diploid strains and the effects of Cadmium treatment on the liver were revealed by histological observation; 2) the liver transcriptomes of triploid crucian carp were obtained and the data was analyzed; 3) 14 genes related to algae and sediments stress were screened from transcriptome data, and the expression of these genes was verified by Quantitative real-time PCR. The results were consistent with the gene results in transcriptome data; 4) Quantitative real-time PCR and enzyme activity analysis all confirmed triploid crucian carp resistance to Cadmium stress was regulated by both oxidative stress and ER stress responses; 5) We found that IRE-1 and PERK, not ATF- 6, were involved in the enhance Cadmium resistance of triploid crucian carp. CONCLUSION: these studies demonstrate that triploid crucian carp have strong resistance to Cadmium.

Persistence and Transcription of Paternal mtDNA Dependent on the Delivery Strategy Rather than Mitochondria Source in Fish Embryos.

BACKGROUND/AIMS: Mitochondria (MT) and mitochondrial DNA (mtDNA) show maternal inheritance in most eukaryotic organisms; the sperm mtDNA is usually delivered to the egg during fertilization and then rapidly eliminated to avoid heteroplasmy, which can affect embryogenesis. In our previous study, fertilization-delivered sperm mtDNA exhibited late elimination and transcriptional quiescence in cyprinid fish embryos. However, the mechanisms underlying elimination and transcriptional quiescence of paternal mtDNA are unclear. METHODS: Goldfish and zebrafish were used to investigate the fate of mtDNAs with different parental origins delivered by fertilization or microinjection in embryos. Goldfish MT from heart, liver and spermatozoa were microinjected into zebrafish zygotes, respectively. Specific PCR primers were designed so that the amplicons have different sizes to characterize goldfish and zebrafish cytb genes or their cDNAs. RESULTS: The MT injection-delivered paternal mtDNA from sperm, as well as those from the heart and liver, was capable of persistence and transcription until birth, in contrast to the disappearance and transcriptional quiescence at the heartbeat stage of fertilization-delivered sperm mtDNA. In addition, the exogenous MT-injected zebrafish embryos have normal morphology during embryonic development. CONCLUSIONS: The fate of paternal mtDNA in fishes is dependent on the delivery strategy rather than the MT source, suggesting that the presence of sperm factor(s) is responsible for elimination and transcriptional quiescence of fertilization-delivered sperm mtDNA. These findings provide insights into the mechanisms underlying paternal mtDNA fate and heteroplasmy in cyprinid fishes.

Comparative Transcriptome and DNA methylation analyses of the molecular mechanisms underlying skin color variations in Crucian carp (Carassius carassius L.).

BACKGROUND: Crucian carp is a popular ornamental strain in Asia with variants in body color. To further explore the genetic mechanisms underlying gray and red body color formation in crucian carp, the skin transcriptomes and partial DNA methylation sites were obtained from red crucian carp (RCC) and white crucian carp (WCC). Here, we show significant differences in mRNA expression and DNA methylation sites between skin tissues of RCC and WCC. RESULTS: Totals of 3434 and 3683 unigenes had significantly lower and higher expression in WCC, respectively, compared with unigenes expressed in RCC. Some potential genes for body color development were further identified by quantitative polymerase chain reaction, such as mitfa, tyr, tyrp1, and dct, which were down-regulated, and foxd3, hpda, ptps, and gch1, which were up-regulated. A KEGG pathway analysis indicated that the differentially expressed genes were mainly related to mitogen activated protein kinase (MAPK), Wnt, cell cycle, and endocytosis signaling pathways, as well as variations in melanogenesis in crucian carp. In addition, some differentially expressed DNA methylation site genes were related to pigmentation, including mitfa, tyr, dct, foxd3, and hpda. The differentially expressed DNA methylation sites were mainly involved in signaling pathways, including MAPK, cAMP, endocytosis, melanogenesis, and Hippo. CONCLUSIONS: Our study provides the results of comparative transcriptome and DNA methylation analyses between RCC and WCC skin tissues and reveals that the molecular mechanism of body color variation in crucian carp is strongly related to disruptions in gene expression and DNA methylation during pigmentation.

Comparative transcriptome analysis of molecular mechanism underlying gray-to-red body color formation in red crucian carp (Carassius auratus, red var.).

Red crucian carp (Carassius auratus red var.) is an ornamental fish with vivid red/orange color. It has been found that the adult body color of this strain forms a gray-to-red change. In this study, skin transcriptomes of red crucian carp are first obtained for three different stages of body color development, named by gray-color (GC), color-variation (CV), and red-color (RC) stages, respectively. From the skins of GC, CV, and RC, 103,229; 108,208; and 120,184 transcripts have been identified, respectively. Bioinformatics analysis reveals that 2483, 2967, and 4473 unigenes are differentially expressed between CV and GC, RC and CV, and RC and GC, respectively. A part of the differentially expressed genes (DEGs) are involved in the signaling pathway of pigment synthesis, such as the melanogenesis genes (Mitfa, Pax3a, Foxd3, Mc1r, Asip); tyrosine metabolism genes (Tyr, Dct, Tyrp1, Silva, Tat, Hpda); and pteridine metabolism genes (Gch, Xdh, Ptps, Tc). According to the data of transcriptome and quantitative PCR, the expression of Mitfa and its regulated genes which include the genes of Tyr, Tyrp1, Dct, Tfe3a, and Baxα, decreases with gray-to-red change. It is suggested that Mitfa and some genes, being related to melanin synthesis or melanophore development, are closely related to the gray-to-red body color transformation in the red crucian carp. Furthermore, the DEGs of cell apoptosis and autophagy pathway, such as Tfe3a, Baxα, Hsp70, Beclin1, Lc3, Atg9a, and Atg4a, might be involved in the melanocytes fade away of juvenile fish. These results shed light on the regulation mechanism of gray-to-red body color transformation in red crucian carp, and are helpful to the selective breeding of ornamental fish strains.

Mitochondrial complex I deficiency leads to the retardation of early embryonic development in Ndufs4 knockout mice.

BACKGROUND: The NDUFS4 gene encodes an 18-kD subunit of mitochondria complex I, and mutations in this gene lead to the development of a severe neurodegenerative disease called Leigh syndrome (LS) in humans. To investigate the disease phenotypes and molecular mechanisms of Leigh syndrome, the Ndufs4 knockout (KO) mouse has been widely used as a novel animal model. Because the homozygotes cannot survive beyond child-bearing age, whether Ndufs4 and mitochondrial complex I influence early embryonic development remains unknown. In our study, we attempted to investigate embryonic development in Ndufs4 KO mice, which can be regarded as a Leigh disease model and were created through the CRISPR (clustered regularly interspaced short palindromic repeat) and Cas9 (CRISPR associated)-mediated genome editing system. METHODS: We first designed a single guide RNA (sgRNA) targeting exon 2 of Ndufs4 to delete the NDUFS4 protein in mouse embryos to mimic Leigh syndrome. Then, we described the phenotypes of our mouse model by forced swimming and the open-field test as well as by assessing other behavioral characteristics. Intracytoplasmic sperm injection (ICSI) was performed to obtain KO embryos to test the influence of NDUFS4 deletion on early embryonic development. RESULTS: In this study, we first generated Ndufs4 KO mice with physical and behavioral phenotypes similar to Leigh syndrome using the CRISPR/Cas9 system. The low developmental rate of KO embryos that were derived from knockout gametes indicated that the absence of NDUFS4 impaired the development of preimplantation embryos. DISCUSSION: In this paper, we first obtained Ndufs4 KO mice that could mimic Leigh syndrome using the CRISPR/Cas9 system. Then, we identified the role of NDUFS4 in early embryonic development, shedding light on its roles in the respiratory chain and fertility. Our model provides a useful tool with which to investigate the function of Ndufs4. Although the pathological mechanisms of the disease need to be discovered, it helps to understand the pathogenesis of NDUFS4 deficiency in mice and its effects on human diseases.

A light-colored region of caudal fin: a niche of melanocyte progenitors in crucian carp (Cyprinus carpio L.).

Melanocyte stem cells are a population of immature cells which sustain the self-renewal and replenish the differentiated melanocytes. In this research, a light-colored region (LCR) is observed at the heel of caudal fin in juvenile crucian carp. By cutting off the caudal fin, the operated caudal fin can regenerate in accordance with the original pigment pattern from the retained LCR. As markers of stem cells, Oct4 and Sox2 have been found to be highly expressed in the LCR as well as Mitfa, a label of the melanoblasts. In vitro, Mitfa+ melanoblasts are observed in the cells which are derived from the LCR and transfected with Mitfa-EGFP reporter by using Tol2 transposon system. Furthermore, by real-time qPCR, it is shown that the level of sox2 mRNA is gradually decreased from the LCR to proximal and distal caudal fin, and that of mitfa mRNA in the proximal caudal fin (PCF) is higher than that in the LCR, while it is the lowest in the distal caudal fin. Hence, we propose that the LCR is a pigment progenitor niche, sending melanocytes to the distal of caudal fin, which gradually emerges as caudal fin grow. We reveal that the LCR of caudal fin might be a niche of pigment progenitors, and contribute to pigment-producing stem cells in crucian carp.

Autotetraploid cell line induced by SP600125 from crucian carp and its developmental potentiality.

Polyploidy has many advantages over diploidy, such as rapid growth, sterility, and disease resistance, and has been extensively applied in agriculture and aquaculture. Though generation of new polyploids via polyploidization has been achieved in plants by different ways, it is comparatively rare in animals. In this article, by a chemical compound, SP600125, polyploidization is induced in fish cells in vitro, and a stable autotetraploid cell line has been generated from diploid fibroblast cells of crucian carp. As a c-Jun N-terminal kinase (Jnk) inhibitor, SP600125 does not function during the induction process of polyploidization. Instead, the p53 signal pathway might be involved. Using the SP600125-induced tetraploid cells and eggs of crucian carp as the donors and recipients, respectively, nuclear transplantation was conducted such that tetraploid embryos were obtained. It suggests that combining polyploidization and the somatic cell nuclear transfer technique (SCNT) is an efficient way to generate polyploidy, and the presented method in this research for generating the tetraploid fish from diploid fish can provide a useful platform for polyploid breeding.