Establishment of cell-lines stably expressing recombinant Japanese eel follicle-stimulating hormone and luteinizing hormone using CHO-DG44 cells: fully induced ovarian development at different modes.

The gonadotropins (Gth), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), play central roles in gametogenesis in vertebrates. However, available information on their differential actions in teleost, especially in vivo, is insufficient. In this study, we established stable CHO-DG44 cell lines expressing long-lasting recombinant Japanese eel Fsh and Lh with extra O-glycosylation sites (Fsh-hCTP and Lh-hCTP), which were produced in abundance. Immature female eels received weekly intraperitoneal injections of Gths. Fsh-hCTP induced the entire ovarian development by 8 weeks from the beginning of injection; thus, the ovaries of most fish were at the migratory nucleus stage while the same stage was observed in eels after 4 weeks in the Lh-hCTP-treated group. In contrast, all pretreated and saline-injected eels were in the pre-vitellogenic stage. Gonadosomatic indices in the Fsh-hCTP-treated group were significantly higher than those in the Lh-hCTP group at the migratory nucleus stage because of the significantly higher frequency of advanced ovarian follicles. Ovarian mRNA levels of genes related to E2 production (cyp11a1, cyp17a1, cyp19a1, hsd3b, fshr, and lhr) were measured using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). All genes were induced by both Fsh-hCTP and Lh-hCTP, with a peak at either the mid- or late vitellogenic stages. Transcript abundance of cyp19a1 and fshr in the Lh-hCTP group were significantly higher than those in the Fsh-hCTP group, whereas no difference in the expression of other genes was observed between the groups. Fluctuations in serum levels of sex steroid hormones (estradiol-17ß, 11-ketotestosterone, and testosterone) in female eels were comparable in the Fsh-hCTP and Lh-hCTP groups, thus increasing toward the maturational phase. Furthermore, the fecundity of the eels induced to mature by Fsh-hCTP was significantly higher than that induced by Lh-hCTP. These findings indicate that Fsh and Lh can induce ovarian development in distinctively different modes in the Japanese eel.

Maternal transcripts in good and poor quality eggs from Japanese eel, Anguilla japonica-their identification by large-scale quantitative analysis.

Our understanding of maternal control of development in vertebrates remains incomplete. In this study, we investigated levels of maternal transcripts in good and poor quality eggs from artificially matured Japanese eel, using RNA-Seq and quantitative polymerase chain reaction (qPCR), to identify candidate maternal transcripts related to development. De novo assembly or mapping of reads to the eel draft genome yielded 619,029 contigs and 85,906 transcripts, respectively; normalized read counts to these assemblies were calculated using reads (RPKM) or fragments (FPKM) per kilobase of transcript per million mapped reads. In silico screening identified 1,594 contigs and 150 transcripts with lower RPKM or FPKM in poor than in good quality eggs, 245 contigs, and 85 transcripts of which could be annotated by BLASTx, respectively. From selected contigs or transcripts, six genes (dnajb4, gnpat, card14, pdp1, fcgbp, ttn) had significantly lower messenger RNA levels in poor than in good quality eggs by qPCR. Multiple regression analysis showed that five genes (gnpat, b4galnt1, acsl6, rtkn, trim24) significantly correlated with hatchability. Taken together, 10 genes were identified as candidate maternal transcripts, regulating development in Japanese eel. Our results contribute to understanding the molecular basis for maternal control of development in vertebrates.

Reduced scytonemin isolated from Nostoc commune induces autophagic cell death in human T-lymphoid cell line Jurkat cells.

Nostoc commune is a terrestrial benthic blue-green alga that often forms an extended mucilaginous layer on the soil, accumulates on stones and mud in aquatic environments. Reduced-scytonemin (R-scy), isolated from N. commune Vaucher, has been shown to suppress the human T-lymphoid Jurkat cell growth. To reveal the mechanisms underlying the R-scy-mediated inhibition of Jurkat cell growth, we examined cell morphology, DNA fragmentation, and microtubule-associated protein light chain 3 (LC3) modification in these cells. We observed multiple vacuoles as well as the conversion of LC3-I to LC3-II in R-scy-treated cells. These results suggest that the R-scy induced Jurkat cell growth inhibition is attributable to the induction of type II programmed cell death (PCD II; autophagic cell death or autophagy). We further examined the mechanisms underlying R-scy-induced PCDII. The cells treated with R-scy produced large amounts of reactive oxygen species (ROS), leading to the induction of mitochondrial dysfunction. However, the elimination of R-scy-induced ROS by treatment with N-acetyl-L-cysteine (NAC) markedly opposed R-scy-induced PCDII. Based on these results, we conclude that ROS formation plays a critical role in R-scy-induced PCDII.

Hatching enzyme of Japanese eel Anguilla japonica and the possible evolution of the egg envelope digestion mechanism.

We purified eel hatching enzyme (EHE) from the hatching liquid of Japanese eel Anguilla japonica belonging to Elopomorpha to a single band on SDS/PAGE. TOF-MS analysis revealed that the purified EHE contained several isozymes with similar molecular masses. Comparison of the egg envelope digestion specificities of the purified EHE and of recombinant EHE4, one of the EHE isozymes, suggested that the isozymes contained in the purified EHE were functionally the same in terms of egg envelope digestion. By electron microscopy, the egg envelope became swollen after treatment with the purified EHE. The EHE cleavage sites on the zona pellucida (ZP) protein of the egg envelope were located in the N-terminal repeat regions. In previous phylogenetic analysis, we suggested that fishes included in Elopomorpha, as basal teleosts, possess a single type of hatching enzyme genes, and that fishes in Otocephala and Euteleostei gain two types of hatching enzyme genes called clade I and II genes by duplication. Further, the clade I enzymes, zebrafish hatching enzyme (ZHE1) and medaka high choriolytic enzyme (HCE), swell the egg envelope by cleaving the N-terminal regions of ZP proteins, while the clade II enzyme, medaka low choriolytic enzyme (LCE), solubilizes the swollen envelope by cleaving the site at the middle region on the ZP domain. In this evolutionary scenario, our findings support that hatching of Japanese eel conserves the ancestral mechanism of fish egg envelope digestion. The clade I enzymes inherit the ancestral enzyme function, and the clade II enzymes gain a new function during evolution to Otocephala and Euteleostei.