Sequencing of the black rockfish chromosomal genome provides insight into sperm storage in the female ovary.
ABSTRACT: Black rockfish (Sebastes schlegelii) is an economically important viviparous marine teleost in Japan, Korea, and China. It is characterized by internal fertilization, long-term sperm storage in the female ovary, and a high abortion rate. For better understanding the mechanism of fertilization and gestation, it is essential to establish a reference genome for viviparous teleosts. Herein, we used a combination of Pacific Biosciences sequel, Illumina sequencing platforms, 10× Genomics, and Hi-C technology to obtain a genome assembly size of 848.31?Mb comprising 24 chromosomes, and contig and scaffold N50 lengths of 2.96 and 35.63?Mb, respectively. We predicted 39.98% repetitive elements, and 26,979 protein-coding genes. S. schlegelii diverged from Gasterosteus aculeatus ?32.1-56.8 million years ago. Furthermore, sperm remained viable within the ovary for up to 6?months. The glucose transporter SLC2 showed significantly positive genomic selection, and carbohydrate metabolism-related KEGG pathways were significantly up-regulated in ovaries after copulation. In vitro suppression of glycolysis with sodium iodoacetate reduced sperm longevity significantly. The results indicated the importance of carbohydrates in maintaining sperm survivability. Decoding the S. schlegelii genome not only provides new insights into sperm storage; additionally, it is highly valuable for marine researchers and reproduction biologists.
Project description:Here we report Illumina-based whole genome sequencing of three rockfish species of Sebastes in northwest Pacific. The whole genomic DNA was used to prepare 350-bp pair-end libraries and the high-throughput sequencing yielded 128.5, 137.5, and 124.8 million mapped reads corresponding to 38.54, 41.26, and 37.43?Gb sequence data for S. schlegelii, S. koreanus, and S. nudus, respectively. The k-mer analyses revealed genome sizes were 846.4, 832.5, and 813.1?Mb and the sequencing coverages were 45×, 49×, and 46× for three rockfish, respectively. Comparative genomic analyses identified 46,624 genome-wide single nucleotide polymorphisms (SNPs). Phylogenetic analysis revealed closer relationships of the three species, compared to other six rockfish species. Demographic analysis identified contrasting changes between S. schlegelii and other two species, suggesting drastically different response to climate changes. The reported genome data in this study are valuable for further studies on comparative genomics and evolutionary biology of rockfish species.
Project description:Germ cells play a key role in gonad development. As precursors, primordial germ cells (PGCs) are particularly important for germline formation. However, the origination and migration patterns of PGCs are poorly studied in marine fish, especially for viviparous economic species. The <i>vasa</i> gene has been widely used as a germ cell marker to identify a germline because <i>vasa</i> RNA is a component of germ plasm. In this study, we described the expression pattern of black rockfish (<i>Sebastes schlegelii</i>) <i>vasa</i> (<i>Ssvas</i>) in gonadal formation and development by <i>in situ</i> hybridization. The results showed that <i>Ssvas</i> failed in localization at the cleavage furrows until the late gastrula stage, when PGCs appeared and migrated to the genital ridge and formed elongated gonadal primordia at 10 days after birth. This study firstly revealed the PGCs origination and migration characteristics in viviparous marine fish. Furthermore, we microinjected chimeric mRNA containing EGFP and the 3'untranslated region (3'UTR) of <i>Ssvas</i> into zebrafish (<i>Danio rerio</i>) and marine medaka (<i>Oryzias melastigma</i>) fertilized eggs for tracing PGCs. We found that, although <i>Sebastes schlegelii</i> lacked early localization, similar to red seabream (<i>Pagrus major</i>) and marine medaka, only the 3'UTR of <i>Ssvas vasa</i> 3'UTR of black rockfish was able to label both zebrafish and marine medaka PGCs. In comparison with other three Euteleostei species, besides some basal motifs, black rockfish had three specific motifs of M10, M12, and M19 just presented in zebrafish, which might play an important role in labeling zebrafish PGCs. These results will promote germ cell manipulation technology development and facilitate artificial reproduction regulation in aquaculture.
Project description:The recent miniaturization of acoustic tracking devices has allowed fishery managers and scientists to collect spatial and temporal data for sustainable fishery management. The spatial and temporal dimensions of fish behavior (movement and/or vertical migrations) are particularly relevant for rockfishes (Sebastes spp.) because most rockfish species are long-lived and have high site fidelity, increasing their vulnerability to overexploitation. In this study, we describe the short-term (with a tracking period of up to 46 d) spatial behavior, as determined by acoustic tracking, of the black rockfish Sebastes schlegelii, a species subject to overexploitation in the Yellow Sea of China. The average residence index (the ratio of detected days to the total period from release to the last detection) in the study area was 0.92 ± 0.13, and most of the tagged fish were detected by only one region of the acoustic receiver array, suggesting relatively high site fidelity to the study area. Acoustic tracking also suggested that this species is more frequently detected during the day than at night in our study area. However, the diel detection periodicity (24 h) was only evident for certain periods of the tracking time, as revealed by a continuous wavelet transform. The habitat selection index of tagged S. schlegelii suggested that S. schlegelii preferred natural reefs, mixed sand/artificial reef bottoms and mixed bottoms of boulder, cobble, gravel and artificial reefs. The preference of this species for the artificial reefs that were recently deployed in the study area suggests that artificial seascapes may be effective management tools to attract individuals. The vertical movement of tagged S. schlegelii was mostly characterized by bottom dwelling behavior, and there was high individual variability in the vertical migration pattern. Our results have important implications for S. schlegelii catchability, the implementation of marine protected areas, and the identification of key species habitats, and our study provides novel information for future studies on the sustainability of this important marine resource in eastern China.
Project description:Mechanisms regulating sperm motility activation are generally known in oviparous fishes, but are poorly understood in viviparous species. The mechanism of osmotic-shock induced signaling for oviparous fishes is not suitable for viviparous fishes which activate sperm motility within an isotonic environment. In addition, the presence of sperm bundles in viviparous fishes further complicates study of sperm activation mechanisms. The goal of this study was to establish methodologies to detect intracellular Ca2+ signals from sperm cells within bundles, and to investigate the signaling mechanism of sperm activation of viviparous fish using Redtail Splitfin (Xenotoca eiseni) as a model. Motility was assessed by classification of bundle dissociation and computer-assisted sperm analysis, and intracellular Ca2+ was assessed using the fluorescent probe Fura-2 AM. Bundle dissociation and sperm motility increased with extracellular Ca2+ and pH levels. Intracellular Ca2+ signals were detected from sperm within bundles, and increased significantly with extracellular Ca2+ and pH levels. Major channel blockers known to inhibit Ca2+ influx (NiCl2, ruthenium red, GdCl3, SKF-96365, nimodipine, verapamil, methoxyverapamil, mibefradil, NNC 55-0396, ?-Conotoxin MVIIC, bepridil, and 2-APB) failed to inhibit Ca2+ influx, except for CdCl2, which partially inhibited the influx. We propose a novel mechanism for motility regulation of fish sperm: an alkaline environment in the female reproductive tract opens Ca2+ channels in the sperm plasma membrane without osmotic shock, and the Ca2+ influx functions as a second messenger to activate motor proteins controlling flagella movement.
Project description:This paper presents data associated with the research article entitled "Targeted downregulation of s36 protein unearths its cardinal role in chorion biogenesis and architecture during <i>Drosophila melanogaster</i> oogenesis" . <i>Drosophila</i> chorion is produced by epithelial follicle cells and one of its functional serving role is egg fertilization through the micropyle, a specialized narrow channel at the anterior tip of the egg . Sperm entry during fertilization is necessary for the egg to complete meiosis . <i>D. melanogaster</i> flies being characterized by severe downregulation of the s36 chorionic protein, specifically in the follicle-cell compartment of their ovary, appear with impaired fly fertility (Velentzas et al., 2016) . In an effort to further investigate whether the observed infertility in the s36-targeted flies derives from a fertilization failure, such as the inability of sperm to pass through egg?s micropyle, we mated females carrying s36-depleted ovaries with males expressing the GFP protein either in their sperm tails, or in both their sperm tails and sperm heads.
Project description:<h4>Background</h4>The black rockfish (Sebastes schlegelii) has an ovoviviparous reproductive pattern and long-term sperm storage, resulting in asynchronous gonadal development between the sexes. However, the comprehensive understanding of gonadal development in black rockfish has not yet been achieved. Here, we studied gonadal development and germ cell renewal using histology and RNA-seq.<h4>Results</h4>In this study, RNA-seq was performed on testes and ovaries to characterize key pathways and genes that are active during development and gamete maturation in black rockfish. Differentially expressed genes (DEGs) were identified and annotated in 4 comparisons (F_III vs. F_IV, F_IV vs. F_V, M_III vs. M_IV and M_IV vs. M_V). Based on analysis of DEGs enriched in the testis, 11 and 14 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were mapped to the M_III vs. M_IV group and the M_IV vs. M_V group, respectively. DEGs in ovarian development were also classified into 10 groups according to their biological functions. The expression patterns of the selected genes determined by qPCR were significantly correlated with the RNA-Seq results, supporting the reliability and accuracy of the RNA-Seq analysis. E<sub>2</sub> levels showed down regulation from previtellogenesis to mature stage in female and T level showed down regulation from spermatogenesis to regressed stage in the male.<h4>Conclusions</h4>The categories "intercellular interaction and cytoskeleton", "molecule amplification" and "repair in the cell cycle" were revealed to be crucial in testis development and spermatogenesis, as was the biosynthesis of a series of metabolites. Our results provide comprehensive insight into black rockfish gonadal development and provide a basis for further study of reproductive physiology and molecular biology in ovoviviparity teleosts.
Project description:Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B-SLLP1 as a pair of novel sperm-egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.
Project description:Oviducal glands (OGs) are distinct expanded regions of the anterior portion of the oviduct, commonly found in chondrichthyans, which play a key role in the production of the egg in-vestments and in the female sperm storage (FSS). The FSS phenomenon has implications for understanding the reproductive ecology and management of exploited populations, but little information is available on its taxonomic extent. For the first time, mature OGs from three lecithotrophic oviparous and four yolk-sac viviparous species, all considered at risk from the fishing impacts in the central western Mediterranean Sea, were examined using light microscopy. The OG microanatomy, whose morphology is generally conserved in all species, shows differences within the two reproductive modalities. Oviparous species show a more developed baffle zone in respect to viviparous ones because of the production of different egg envelopes produced. Among oviparous species, <i>Raja polystigma</i> and <i>Chimaera monstrosa</i> show presence of sperm, but not sperm storage as observed, instead, in <i>Galeus melastomus</i> and in all the viviparous sharks, which preserve sperm inside of specialized structures in the terminal zone.
Project description:We report the complete genome sequence of the virulent <i>Aeromonas salmonicida</i> subsp. <i>masoucida</i> strain BR19001YR, isolated from diseased black rockfish (<i>Sebastes schlegelii</i>). Sequencing of the circular chromosome and three plasmids using the PacBio and Illumina platforms yielded 4,982,192 bp with a 58.24% G+C content.
Project description:Sperm samples used on fertilization strongly influence the in vitro production (IVP) rates. However, sperm traits behind this effect are not stated consistently until now. This study aimed to evaluate the isolated and combined effect of some sperm traits (MB: total motility before Percoll® gradient, MA: total motility after Percoll® gradient, AI: acrosome integrity, MI: membrane integrity, MP: mitochondrial membrane potential, and CR: chromatin resistance) on IVP rates. This is the first study focusing on the isolated effect of distinct traits. For this purpose, the experiment was divided in three steps. In first step, to study behavior of traits sperm samples (n = 63 batches) were analyzed and ranked based on each trait. In second step, samples ranked were selected from target ranks regions and allocated in groups of four to five batches, creating Higher and Lower groups, according to two different approaches. One aimed to form groups that differed to all sperm traits simultaneously (effect of combined traits). The other aimed to form groups that differed only to a single sperm trait while no differences were observed for the remaining traits (effect of each isolated trait). In third step, for each group successfully formed in step 2, sperm samples were individually and prospectively used for IVP. Cleavage, embryo development and blastocyst rates were recorded and compared between Higher and Lower of respective trait groups. Surprisingly, evaluation of isolated effects revealed that lower levels of MB, AI and MP resulted in higher embryo development and blastocyst rates (p<0.05), which was not observed on cleavage rate. We conclude that sperm traits strongly influence embryo development after in vitro fertilization (IVF), affecting the zygote competence to achieve blastocyst stage. Individually, levels of MB, AI or MP could be some of the key traits that may define IVP efficiency on current systems of embryo production.