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.

Molecular characterization and expression pattern of a germ cell marker gene dnd in gibel carp (Carassius gibelio).

As a germ cell marker gene, Dead end (dnd) has been identified and characterized in many vertebrates. Recently, we created a complete germ cell-depleted gonad model by the dnd-specific morpholino-mediated knockdown approach, and revealed sex-biased gene expression alteration through utilizing unisexual gynogenetic superiority in polyploid gibel carp. However, dnd and its expression pattern are still unclear in the gibel carp. In this study, we further analyzed molecular characterization of gibel carp dnd and its dynamic expression pattern during gametogenesis and embryogenesis. Similar to other homologs in vertebrates, gibel carp dnd contains a conserved RRM motif and five other motifs, and is highly evolutionary conserved in genomic organization and neighborhood gene synteny. RT-PCR and Western blot analyses showed its gonad-specific expression intensively in testis and ovary. Section in situ hybridization (SISH) and immunofluorescence localization revealed its dynamic expression pattern specific to oogenic cells and spermatogenetic cells during oogenesis and spermatogenesis. Moreover, its temporal and spatial distribution specific to PGCs were also demonstrated by RT-PCR and whole mount in situ hybridization (WISH) during embryogenesis. Therefore, gibel carp Dnd is a conserved germ cell marker during gametogenesis, and its maternal transcript is also a useful marker for tracing PGC specification and migration.

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.

[Chromosomal localization of rice field eel Hox genes by PRINS].

The genome duplication and chromosome rearrangement are two kinds of evolution models at the chromosome level during the evolution of vertebrate genome. And Hox genes are the powerful proves to support the evolution theory of genome duplication, which has been found recently. In this study, the chromosomal localization of rice field eel Hox genes has been carried out by PRINS. The mapping results indicated that 6 Hox clusters might exist in rice field eel genome, and these clusters were localized on chromosome 1, 2, 3, 6, 8, 10 and at the position of 28.24 +/- 2.88, 4.55 +/- 1.39, 13.89 +/- 2.03, 74.32 +/- 1.86, 38.03 +/- 2.41, 58.18 +/- 2.05 from the centromere respectively. The mapping results that Hox genes were localized on chromosome 1, 3, 6 and 10 in the study are corresponding to that by chromosome microdissection. The chromosomal localization of rice field eel Hox genes will help us to discover the origin and evolution of rice field eel chromosomes, and provide cellular genetic proves of this special species to support the evolution theory of genome duplication.

[Construction and homologous detection of a DNA plasmid library specific for Z chromosome of quail].

A simple method was used to adapt a standard light microscope for the collection of quail Z chromosomes from mitotic-metaphase spreads. The microisolated chromosomes were subjected to proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed PCR (DOP-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis, and smears of DNA revealed that ranged in size from 200-1 400 bp, without any evidence of preferential amplification. The second-round PCR products were cloned into pBluescript plasmids to construct a Z chromosome-specific DNA library. The size range of the cloned inserts was 200-1 400 bp. Using inserted fragments from the library as probes, chromosome painting was performed on quail chromosomes. The results showed that Z chromosomes of quail were completely covered by strong signals and there were little signals on other chromosomes. It was indicated that inserted DNA of the library was specific to the Z chromosome of quail. The library can be used as chromosome painting probe to detect conserved syntenic groups on the chromosomes of other related species and study mechanisms of sex-chromosomes evolution in birds.