Production of functional sperm from in vitro-cultured premeiotic spermatogonia in a marine fish.

In vitro production of functional gametes can revolutionize reproduction by reducing generation intervals and accelerating genetic breeding in aquaculture, especially in fish with relatively long generations. Nevertheless, functional sperm production from in vitro-cultured spermatogonia remains a challenge in most aquaculture fish. In this study, we isolated and characterized premeiotic spermatogonia from marine four-eyed sleepers ( Bostrychus sinensis), which are prone to ovotesticular or sterile testicular development, and induced the differentiation of the spermatogonia into flagellated sperm in a three-dimensional (3D) culture system. Artificial insemination indicated that the in vitro-derived sperm were capable of fertilizing mature oocytes to develop into normal larvae. Furthermore, melatonin significantly promoted spermatogonia proliferation and differentiation through the ERK1/2 signaling pathway, and thus increased the efficiency in functional sperm production. The 3D culture system and resulting functional sperm hold great promise for improving the genetic breeding of aquaculture fish.

Capsid protein from red-spotted grouper nervous necrosis virus induces incomplete autophagy by inactivating the HSP90ab1-AKT-MTOR pathway.

As a highly important fish virus, nervous necrosis virus (NNV) has caused severe economic losses to the aquaculture industry worldwide. Autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of several viruses. Although NNV can induce autophagy to facilitate infection in grouper fish spleen cells, how it initiates and mediates autophagy pathways during the initial stage of infection is still unclear. Here, we found that red-spotted grouper NNV (RGNNV) induced autophagosome formation in two fish cell lines at 1.5 and 3 h post infection, indicating that autophagy is activated upon entry of RGNNV. Moreover, autophagic detection showed that RGNNV entry induced incomplete autophagy by impairing the fusion of autophagosomes with lysosomes. Further investigation revealed that binding of the RGNNV capsid protein (CP) to the Lateolabrax japonicus heat shock protein HSP90ab1 (LjHSP90ab1), a cell surface receptor of RGNNV, contributed to RGNNV invasion-induced autophagy. Finally, we found that CP blocked the interaction of L. japonicus protein kinase B (AKT) with LjHSP90ab1 by competitively binding the NM domain of LjHSP90ab1 to inhibit the AKT-mechanistic target of the rapamycin (MTOR) pathway. This study provides novel insight into the relationship between NNV receptors and autophagy, which may help clarify the pathogenesis of NNV.

Identification of MicroRNAs in Zebrafish Spermatozoa.

MicroRNAs (miRNAs) participate in almost all biological processes. Plenty of evidences show that some testis- or spermatozoa-specific miRNAs play crucial roles in the process of gonad and germ cell development. In this study, the spermatozoa miRNA profiles were investigated through a combination of illumina deep sequencing and bioinformatics analysis in zebrafish. Deep sequencing of small RNAs yielded 11,820,680 clean reads. By mapping to the zebrafish genome, we identified 400 novel and 204 known miRNAs that could be grouped into 104 families. Furthermore, we selected the six highest expressions of known miRNAs to detect their expression patterns in different tissues by stem-loop quantitative real-time polymerase chain reaction. We found that among the six miRNAs, dre-miR-202-5p displayed specific and high expression in zebrafish spermatozoa and testis. Fluorescence in situ hybridization analysis indicated that dre-miR-202-5p was predominantly expressed in all kind of germ cells at different spermatogenetic stages, including spermatogonia and spermatozoa, but barely expressed in the germ cells in the ovary. This sex-biased expression pattern suggests that dre-miR-202-5p might be related to spermatogenesis and the functioning of spermatozoa. The identification of miRNAs in zebrafish spermatozoa and germ cells offers new insights into the spermatogenesis and spermatozoa in the teleost and other vertebrates.

Complete Genome Sequence of a Fish Nervous Necrosis Virus Isolated from Sea Perch (Lateolabrax japonicus) in China.

We sequenced and analyzed the complete genome of a fish nervous necrosis virus isolated from diseased sea perch (Lateolabrax japonicus) in Guangdong Province, China. The virus genome contains RNA1 (3,103 bp) and RNA2 (1,433 bp). Phylogenetic analysis shows that the virus belongs to the redspotted grouper nervous necrosis virus genotype of betanodavirus.

A truncated Danio rerio PKZ isoform functionally interacts with eIF2α and inhibits protein synthesis.

A protein kinase containing Z-DNA binding domains (PKZ), which resembles protein kinase R (PKR) in domain organization, was recently discovered to be a member of the eIF2α kinase family in fish. PKR has roles in antiviral immunity through inhibiting protein synthesis and activating NF-κB; therefore, it is thought that PKZ may have a similar role in fish antiviral immunity. In the present study, the roles of two Danio rerio PKZ isoforms (DrPKZ-A and DrPKZ-B) in eIF2α phosphorylation and protein synthesis regulation were explored. DrPKZ-A and DrPKZ-B possess N-terminal Z-DNA binding domains and a conserved eIF2α kinase domain; however, they have domains of differing lengths inserted between kinase subdomains IV and V. DrPKZ-A has an insert domain of 73 amino acids (aa), whereas DrPKZ-B has an insert sequence of only 10 aa, suggesting that DrPKZ-B could be a dysfunctional isoform or could interact with different substrates. Our results show that both DrPKZ-A and DrPKZ-B functionally interact with eIF2α and inhibit protein synthesis, although DrPKZ-B possesses attenuated kinase activity. Our results also show that deletion of the insert in either isoform results in the complete abrogation of kinase activity, suggesting that the insert is critical for PKZ kinase activity. Kinase activity appears to be independent of insert length but may depend on the presence of specific amino acids within the insert domain. Furthermore, the effects of the N-terminal regulatory domain on kinase activity were analyzed. Deletion of the N-terminus results in reduced kinase activity of these isoforms relative to the wild-type forms, indicating that the isolated kinase domain is sufficient for eIF2α phosphorylation and that DrPKZ-A and DrPKZ-B may be regulated in a similar manner. Overall, our results show that DrPKZ-B is a functional kinase in zebrafish and contribute to our understanding of the function of PKZ in fish.

The potential role of microfilaments in host cells for infection with infectious spleen and kidney necrosis virus infection.

BACKGROUND: Infectious spleen and kidney necrosis virus (ISKNV) belongs to the genus Megalocytivirus from the family Iridoviridae. Megalocytivirus causes severe economic losses to tropical freshwater and marine culture industry in Asian countries and is devastating to the mandarin fish farm industry in China particularly. METHODS: We investigated the involvement of microfilaments in the early and late stages of ISKNV infection in MFF-1 cells by selectively perturbing their architecture using well-characterized inhibitors of actin dynamics. The effect of disruption of actin cytoskeleton on ISKNV infection was evaluated by indirect immunofluorescence analysis or real-time quantitative PCR. RESULTS: The depolymerization of the actin filaments with cytochalasin D, cytochalasin B, or latrunculin A reduced ISKNV infection. Furthermore, depolymerization of filamentous actin by inhibitors did not inhibit binding of the virus but affected virus internalization in the early stages of infection. In addition, the depolymerization of actin filaments reduced total ISKNV production in the late stages of ISKNV. CONCLUSIONS: This study demonstrated that ISKNV required an intact actin network during infection. The findings will help us to better understand how iridoviruses exploit the cytoskeleton to facilitate their infection and subsequent disease.

Mandarin fish caveolin 1 interaction with major capsid protein of infectious spleen and kidney necrosis virus and its role in early stages of infection.

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. ISKNV is one of the major agents that cause mortality and economic losses to the freshwater fish culture industry in Asian countries, particularly for mandarin fish (Siniperca chuatsi). In the present study, we report that the interaction of mandarin fish caveolin 1 (mCav-1) with the ISKNV major capsid protein (MCP) was detected by using a virus overlay assay and confirmed by pulldown assay and coimmunoprecipitation. This interaction was independent of the classic caveolin 1 scaffolding domain (CSD), which is responsible for interacting with several signaling proteins and receptors. Confocal immunofluorescence microscopy showed that ISKNV MCP colocalized with mCav-1 in the perinuclear region of virus-infected mandarin fish fry (MFF-1) cells, which appeared as soon as 4 h postinfection. Subcellular fractionation analysis showed that ISKNV MCP was associated with caveolae in the early stages of viral infection. RNA interference silencing of mCav-1 did not change virus-cell binding but efficiently inhibited the entry of virions into the cell. Taken together, these results suggested that mCav-1 plays an important role in the early stages of ISKNV infection.

Infectious spleen and kidney necrosis virus (a fish iridovirus) enters Mandarin fish fry cells via caveola-dependent endocytosis.

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. Megalocytiviruses have been implicated in more than 50 fish species infections and currently threaten the aquaculture industry, causing great economic losses in China, Japan, and Southeast Asia. However, the cellular entry mechanisms of megalocytiviruses remain largely uncharacterized. In this study, the main internalization mechanism of ISKNV was investigated by using mandarin fish fry (MFF-1) cells. The progression of ISKNV infection is slow, and infection is not inhibited when the cells are treated with ammonium chloride (NH(4)Cl), chloroquine, sucrose, and chlorpromazine, which are inhibitors of clathrin-dependent endocytosis. The depletion of cellular cholesterol by methyl-ß-cyclodextrin results in the significant inhibition of ISKNV infection; however, the infection is resumed with cholesterol replenishment. Inhibitors of caveolin-1-involved signaling events, including phorbol 12-myristate 13-acetate (PMA), genistein, and wortmannin, impair ISKNV entry into MFF-1 cells. Moreover, ISKNV entry is dependent on dynamin and the microtubule cytoskeleton. Cofraction analysis of ISKNV and caveolin-1 showed that ISKNV colocates with caveolin-1 during virus infection. These results indicate that ISKNV entry into MFF-1 cells proceeds via classical caveola-mediated endocytosis and is dependent on the microtubules that serve as tracks along which motile cavicles may move via a caveola-caveosome-endoplasmic reticulum (ER) pathway. As a fish iridovirus, ISKNV entry into MFF-1 cells is different from the clathrin-mediated endocytosis of frog virus 3 entry into mammalian cells (BHK-21) at 28°C, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection.

Entry of tiger frog virus (an Iridovirus) into HepG2 cells via a pH-dependent, atypical, caveola-mediated endocytosis pathway.

Tiger frog virus (TFV), in the genus Ranavirus of the family Iridoviridae, causes high mortality of cultured tiger frog tadpoles in China. To explore the cellular entry mechanism of TFV, HepG2 cells were treated with drugs that inhibit the main endocytic pathways. We observed that TFV entry was inhibited by NH(4)Cl, chloroquine, and bafilomycin, which can all elevate the pH of acidic organelles. In contrast, TFV entry was not influenced by chlorpromazine or overexpression of a dominant-negative form of Esp15, which inhibit the assembly of clathrin-coated pits. These results suggested that TFV entry was not associated with clathrin-mediated endocytosis, but was related to the pH of acidic organelles. Subsequently, we found that endocytosis of TFV was dependent on membrane cholesterol and was inhibited by the caveolin-1 scaffolding domain peptide. Dynamin and actin were also required for TFV entry. In addition, TFV virions colocalized with the cholera toxin subunit B, indicating that TFV enters as caveola-internalized cargo into the Golgi complex. Taken together, our results demonstrated that TFV entry occurs by caveola-mediated endocytosis with a pH-dependent step. This atypical caveola-mediated endocytosis is different from the clathrin-mediated endocytosis of frog virus 3 (FV3) by BHK cells, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection in lower vertebrates.

Involvement of caveolin-1 in the Jak-Stat signaling pathway and infectious spleen and kidney necrosis virus infection in mandarin fish (Siniperca chuatsi).

Caveolae, the major source of caveolin-1 protein, are specialized invaginated microdomains of the plasma membrane that act as organizing centers for signaling molecules in the immune system. In the present study, we report the cloning and characterization of caveolin-1 (mCav-1) from mandarin fish (Siniperca chuatsi) and study on the roles of mCav-1 in the fish Jak-Stat signaling pathway and in virus infection. The cDNA sequence of mCav-1 was 707bp in size, encoding a protein of 181 amino acids, which was different from the mammalian protein (178 amino acids). The deduced amino acid sequence of mCav-1 shared similar architecture with vertebrate caveolin-1 proteins, but mCav-1 lacked a phosphorylation site (y14). The major subcellular location of mCav-1 was in the caveolae, where the protein appeared to have major functions. Real-time PCR revealed that the expression of the mandarin fish Mx, IRF-1, SOCS1, and SOCS3 genes involved in the poly(I:C)-induced Jak-Stat signaling pathway was impaired by the mCav-1 scaffolding domain peptide (mSDP). In mandarin fish fry (MFF-1) cells, the protein levels of mCav-1 were markedly up-regulated at 12 and 24h post-infection with ISKNV (infectious spleen and kidney necrosis virus). In addition, ISKNV entry into MFF-1 cells was significantly inhibited by mSDP, and the inhibition was dose-dependent. Thus, ISKNV infection was apparently associated with mCav-1 protein and may utilize the caveolae-related endocytosis pathway. The findings reported here further our understanding of the function of caveolin-1 in the complex signal transduction network in fish immune systems and in the cellular entry mechanism of iridoviruses.

Complete genome sequence of a Megalocytivirus (family Iridoviridae) associated with turbot mortality in China.

BACKGROUND: Turbot reddish body iridovirus (TRBIV) causes serious systemic diseases with high mortality in the cultured turbot, Scophthalmus maximus. We here sequenced and analyzed the complete genome of TRBIV, which was identified in Shandong province, China. RESULTS: The genome of TRBIV is a linear double-stranded DNA of 110,104 base pairs, comprising 55% G + C. Total 115 open reading frames were identified, encoding polypeptides ranging from 40 to 1168 amino acids. Amino acid sequences analysis revealed that 39 of the 115 potential gene products of TRBIV show significant homology to other iridovirus proteins. Phylogenetic analysis of conserved genes indicated that TRBIV is closely related to infectious spleen and kidney necrosis virus (ISKNV), rock bream iridovirus (RBIV), orange-spotted grouper iridovirus (OSGIV), and large yellow croaker iridovirus (LYCIV). The results indicated that TRBIV belongs to the genus Megalocytivirus (family Iridoviridae). CONCLUSIONS: The determination of the genome of TRBIV will provide useful information for comparative study of Megalocytivirus and developing strategies to control outbreaks of TRBIV-induced disease.