Comparative Transcriptome Analysis Reveals Sexually Dimorphic Gene Expression in the Gonads of Brachymystax tsinlingensis Li.

Brachymystax tsinlingensis Li is an endangered cold-water salmonid fish native to China. This study aimed to identify sex-related genes and biological pathways via gonadal transcriptome sequencing of B. tsinlingensis Li. A total of 167,904 unigenes were identified with an average length of 836 bp and an N50 of 1452 bp, of which 84,977 (50.61%) unigenes were successfully annotated in six major databases. Comparative transcriptome analysis identified 22,864 differentially expressed genes (DEGs), of which 17,231 were up-regulated (male-biased genes, mDEGs) and 5633 were down-regulated (female-biased genes, fDEGs). Several DEGs associated with gonadal development were found through Gene Ontology enrichment analysis, such as ccnb1, zp3, bmp15, dmrt1, and psmc3ip. Signaling pathways related to gonadal development were found to be enriched through analysis using the Kyoto Encyclopedia of Genes and Genomes Pathway database, such as genes involves in base excision repair, the notch signaling pathway, neuroactive ligand-receptor interaction, the VEGF signaling pathway, and the estrogen signaling pathway. In addition, mRNA expression levels of 19 DEGs were determined to validate the reliability of the transcriptomic data by quantitative real-time polymerase chain reaction. These results revealed genes and signaling pathways potentially involved in gonadal development in B. tsinlingensis Li and provided basic molecular data for future research on reproductive regulation and breeding of B. tsinlingensis Li.

Chromosome-level genome assembly of the largefin longbarbel catfish (Hemibagrus macropterus).

The largefin longbarbel catfish, Hemibagrus macropterus, is an economically important fish species in southwestern China, with males growing faster than females. This study presents a high-quality chromosome-level genome assembly of the largefin longbarbel catfish, generated by integrating Illumina short reads, PacBio HiFi long reads, and Hi-C data. The assembled genome size was 858.5 Mb, with a contig and scaffold N50 of 5.8 Mb and 28.4 Mb, respectively. A total of 656 contigs were successfully anchored to 30 pseudochromosomes with a BUSCO score of 97.7%, consistent with the number of chromosomes analyzed by karyotype. The genome contained 29.5% repeat sequences, and a predicted total of 26,613 protein-coding genes, of which 25,769 (96.8%) were functionally annotated in different databases. Evolutionary analysis showed that H. macropterus was most closely related to H. wyckioides, with a divergence time of approximately 16.3 million years. Chromosomal syntenic relationships among H. macropterus, H. wyckioides, and Pelteobagrus fulvidraco revealed a one-to-one relationship for most chromosomes, except for break, fission, and inversion of some chromosomes. The first high-quality reference genome will not only provide a valuable genetic resource for the study of sex determination mechanisms and genetic breeding of largefin longbarbel catfish, but also contribute to comparative analyses of genome and chromosome evolution within Siluriformes.

Transcriptome Analyses Reveal Expression Profiles of Morphologically Undifferentiated and Differentiated Gonads of Yangtze Sturgeon Acipenser dabryanus.

Sturgeon is known as a primitive fish with the ZZ/ZW sex determination system and is highly prized for its valuable caviar. Exploring the molecular mechanisms underlying gonadal differentiation would contribute to broadening our knowledge on the genetic regulation of sex differentiation of fish, enabling improved artificial breeding and management of sturgeons. However, the mechanisms are still poorly understood in sturgeons. This study aimed to profile expression patterns between female and male gonads at morphologically undifferentiated and early differentiated stages and identify vital genes involved in gonadal sex differentiation of sturgeons. The sexes of Yangtze sturgeon (Acipenser dabryanus) juveniles were identified via the sex-specific DNA marker and histological observation. Transcriptome analyses were carried out on female and male gonads at 30, 80 and 180 days post-hatching. The results showed that there was a total of 17 overlapped DEGs in the comparison groups of between female and male gonads at the three developmental stages, in which there were three DEGs related to ovarian steroidogenesis, including hsd17b1, foxl2 and cyp19a1. The three DEGs were highly expressed in the female gonads, of which the expression levels were gradually increased with the number of days after hatching. No well-known testis-related genes were found in the overlapped DEGs. Additionally, the expression levels of hsd17b1 and cyp19a1 mRNA were decreased with the knockdown of foxl2 mRNA via siRNA. The results further suggested that foxl2 should play a crucial role in the ovarian differentiation of sturgeons. In conclusion, this study showed that more genes involved in ovarian development than testis development emerged with sexually dimorphic expression during early gonadal sex differentiation, and it provided a preliminary understanding of the molecular regulation on gonadal differentiation of sturgeons.

Dietary Epigallocatechin-3-Gallate (EGCG) Improves Nonspecific Immune Response of Chinese Rice Field Eel (Monopterus albus).

Epigallocatechin-3-gallate (EGCG) has been recognized as a potential additive for aquafeeds due to its beneficial biological functions. In order to evaluate the potential application of EGCG in Chinese rice field eel (Monopterus albus), six isonitrogenous and isolipidic diets containing 0, 25, 50, 100, 200, and 400 mg/kg EGCG were formulated and were fed to Monopterus albus (M. albus) for 9 weeks. The results showed that M. albus fed diets containing 0 and 100 mg/kg EGCG presented higher weight again and specific growth rate than the other groups. Fish fed with 25, 50, and 400 mg/kg EGCG displayed lower whole-body lipid content. Serum aspartate aminotransferase (AST) concentration significantly decreased in EGCG treated groups with the exception of 100 mg/kg group. Hepatic catalase (CAT) activity and glutathione (GSH) concentration decreased as EGCG level increased while malondialdehyde (MDA) concentration showed an opposite trend. EGCG supplementation resulted in a promoted lysozyme (LZM) activity and immunoglobulin M (IgM) level in the liver of M. albus. Furthermore, transcription of three immune related genes including major histocompatibility complex (mhc-2α), hepcidin, and interleukin-8 (il-8) mRNAs was upregulated by EGCG treatment; while transcription of interleukin-6 (il-6) and nuclear factor kappa-B (nf-kb) genes was downregulated. Results also showed a linear relation between EGCG inclusion level and parameters of AST, CAT, GSH, MDA, LZM, IgM, and immune-related genes transcriptions. In summary, it could be suggested that EGCG supplementation enhanced the nonspecific immune response of the Chinese rice field eel. Based on the broken-line regression analysis of IgM, the optimal dietary EGCG supplementation for M. albus was estimated to be 109.81 mg/kg.

The last giants of the Yangtze River: A multidisciplinary picture of what remains of the endemic Chinese sturgeon.

The Chinese sturgeon, an important endemism of the Yangtze River, belongs to 'the most critically endangered group of species' worldwide, with overfishing and habitat destruction being the main drivers towards extinction. Newly obtained microchemical comparisons between animals and water from different river locations revealed a probable shifting of the spawning ground few kilometers downstream compared to the only previously known site, located under the Gezhouba Dam. This offers a glimmer of hope for an adaptive response to habitat perturbation caused by the recently built Three Gorges dam on the Yangtze River. On the other hand, genetic data provide an estimate of about 20 breeders participating in the only significant breeding event of the past 10 years. This warns of a near species extinction forecast if no in situ and ex situ conservation efforts occur promptly. Given these results we propose a list of priority conservation actions that urgently need to be promoted, supported, and put into practice.

Spermatogonia From Cryopreserved Testes of Critically Endangered Chinese Sturgeon Efficiently Colonized and Preferentially Proliferated in the Recipient Gonads of Yangtze Sturgeon.

The critically endangered Chinese sturgeon, Acipenser sinensis, presents late sexual maturity and has a large body size. Germ cell transplantation is a powerful technique for the production of gametes from large-bodied species in closely related recipients with a smaller body size and shorter generation time. To accelerate reproduction of Chinese sturgeon, donor spermatogonia collected from the cryopreserved testes of 3-year-old Chinese sturgeon were intraperitoneally transplanted into 7-8 days post-hatch larvae of Yangtze sturgeon (Acipenser dabryanus) with shorter generation interval. At 2 months post-transplantation (mpt), donor spermatogonia had colonized in the 81.25% of recipient gonads, with average numbers about two times those of endogenous primordial germ cells. Within the next 2 months, the rate of endogenous germ cell division in females (2-3 times) was faster than that in males (once), whereas colonized donor-derived spermatogonia divided about 2-3 times and twice in recipient females and males, respectively. Furthermore, the expression of germ cell-related genes, dazl, dead end, and vasa, in transplanted fish was higher than that in non-transplanted fish, suggesting the incorporation and proliferation donor spermatogonia in recipient. At 18 mpt, donor-derived spermatogonia survived in the 75.00% of recipient gonads. These results showed that the somatic microenvironment of Yangtze sturgeon gonad can support the long-term colonization, proliferation, and survival of xenogeneic germ cells. Thus, this study suggested that small-bodied Yangtze sturgeon is promising recipient as surrogate for Chinese sturgeon gamete production.

Screening and identification of female-specific DNA sequences in octaploid sturgeon using comparative genomics with high-throughput sequencing.

In this study, six candidate female-specific DNA sequences of octaploid Amur sturgeon (Acipenser schrenckii) were identified using comparative genomic approaches with high-throughput sequencing data. Their specificity was confirmed by traditional PCR. Two of these sex-specific sequences were also validated as female-specific in other eight sturgeon species and two hybrid sturgeons. The identified female-specific DNA fragments suggest that the family Acipenseridae has a ZZ/ZW sex-determining system. However, one of the two DNA sequences has been deleted in some sturgeons such as Sterlet sturgeon (Acipenser ruthenus), Beluga (Huso huso) and Kaluga (H. dauricus). The difference of sex-specific sequences among sturgeons indicates that there are different sex-specific regions among species of sturgeon. This study not only provided the sex-specific DNA sequences for management, conservation and studies of sex-determination mechanisms in sturgeons, but also confirmed the capability of the workflow to identify sex-specific DNA sequences in the polyploid species with complex genomes.

The American Paddlefish Genome Provides Novel Insights into Chromosomal Evolution and Bone Mineralization in Early Vertebrates.

Sturgeons and paddlefishes (Acipenseriformes) occupy the basal position of ray-finned fishes, although they have cartilaginous skeletons as in Chondrichthyes. This evolutionary status and their morphological specializations make them a research focus, but their complex genomes (polyploidy and the presence of microchromosomes) bring obstacles and challenges to molecular studies. Here, we generated the first high-quality genome assembly of the American paddlefish (Polyodon spathula) at a chromosome level. Comparative genomic analyses revealed a recent species-specific whole-genome duplication event, and extensive chromosomal changes, including head-to-head fusions of pairs of intact, large ancestral chromosomes within the paddlefish. We also provide an overview of the paddlefish SCPP (secretory calcium-binding phosphoprotein) repertoire that is responsible for tissue mineralization, demonstrating that the earliest flourishing of SCPP members occurred at least before the split between Acipenseriformes and teleosts. In summary, this genome assembly provides a genetic resource for understanding chromosomal evolution in polyploid nonteleost fishes and bone mineralization in early vertebrates.

Cryopreservation of germline stem cells in American paddlefish (Polyodon spathula).

Most sturgeon and paddlefish are critically endangered; therefore, effective measures to conserve these genetic resources are required. Cryopreservation of gonad tissues containing germline stem cells could be an effective strategy for long term preservation and restoration of fish species using germ cell transplantation procedure. The aim of this study was to develop an optimal procedure for long-term cryopreservation of American paddlefish gonads using a slow-freezing method. Through optimization of permeating cryoprotectants, nonpermeating cryoprotectants, and supplementation of proteins, gonad tissues were frozen with a cryomedium containing 1.3 M dimethyl sulfoxide, 0.1 M trehalose, and 10 % fetal bovine serum at a cooling rate of -1 °C/min. This method was also successfully utilized for the cryopreservation of Yangtze sturgeon testes. Viability of gonadal cells isolated from frozen gonads was not different from cells isolated from fresh gonadal tissues, while the number of gonadal cells dissociated from frozen gonads was less. Germline stem cells dissociated from long-term (1 year) cryopreserved gonads were labeled with PKH26 fluorescent dye and intraperitoneally transplanted into larvae of Yangtze sturgeon. The colonization of transplanted germline stem cells was confirmed by the presence of PKH26-labeled donor germline stem cells and donor-derived mtDNA sequence in the recipient gonads, providing evidence that germline stem cells from sturgeon and paddlefish gonads that had been preserved for a long period maintained their functions. The results of present study indicate the procedures used are effective for long-term preservation of critically endangered species within the Acipenseriformes order which can later be regenerated using surrogate broodstock technology.

Assessment of Yangtze sturgeon as recipient for the production of American paddlefish gametes through spermatogonia transplantation.

The Chinese paddlefish (Psephurus gladius), one of the world's largest freshwater fish, was last seen alive in 2003; they are presumed now to be extinct. In fish, germ cell transplantation is currently known as one of the most powerful assisted reproductive technologies for the conservation of endangered species. In the event that a Chinese paddlefish is unexpectedly caught in the near future, we aimed to develop an experimental strategy to produce paddlefish gametes in the gonads of surrogate sturgeon. Spermatogonia were collected from the testes of 2.5-year-old immature male American paddlefish (Polyodon spathula), the species most closely related to the Chinese paddlefish, by Percoll gradient centrifugation, and transplanted into the peritoneal cavity of Yangtze sturgeon (Acipenser dabryanus) larvae at 7-8 days post-hatch. At two months post-transplantation, donor-derived spermatogonia had efficiently colonized in the recipient gonads and proliferated. A PCR analysis developed to detect xenogenic donor-derived mtDNA sequences in recipient gonads revealed that American paddlefish germ cells survived for at least seven months after transplantation in the gonads of Yangtze sturgeon recipients. These results show that the somatic microenvironment of Yangtze sturgeon gonads was able to support the colonization, proliferation, and survival of xenogeneic germ cells from a different taxonomic family. This study provides key information that could lead to future restoration of Chinese paddlefish using germ cell transplantation.

Transcriptome analysis of ovarian maturation in a chondrostei Chinese sturgeon Acipenser sinensis.

Chinese sturgeon (Acipenser sinensis) with an evolutionary history of over 200 million years, has a long lifespan, and an extremely late and asynchronous sexual maturation (8-18 years for males and 14-26 years for females), resulting in the difficulty of mature adult culture. However, little is known about the regulatory mechanisms of the transition among ovarian maturation stages in the Chinese sturgeon. We performed de novo transcriptome sequencing of the Chinese sturgeon at different ovarian maturation stages (FII, FIII, and FIV). The number of differentially expressed genes (DEGs) between FII and FIII/FIV (33,517/34,217) was more than that between FIII and FIV (22,123), suggesting that the transition from FII to FIII/FIV is more important than that from FIII to FIV for ovarian maturation. The number of upregulated genes was more than that of the downregulated genes, suggesting that increased gene expression was involved in the transition from FII to FIII/FIV. The representative pathways of DEGs were steroid biosynthesis, fatty acid biosynthesis, fatty acid elongation, glycerolipid metabolism, biosynthesis of unsaturated fatty acid, and α-linolenic acid metabolism. The differential expressions from the transcriptome sequencing were validated with real-time reverse-transcription polymerase chain reaction. We also found 13 genes in sexual development, female sex determination, gonadal development, ovarian maturation, ovarian follicle development, and oocyte development pathways, which were differently expressed among fish at FII, FIII, and FIV. We suggest that enhanced synthesis of steroid, unsaturated fatty acid, and α-linolenic acid may contribute to ovarian maturation of the Chinese sturgeon. These potential determinants provide a glimpse of the molecular architecture of ovary development in sturgeons.

Draft Genome and Complete Hox-Cluster Characterization of the Sterlet (Acipenser ruthenus).

Background: Sturgeons (Chondrostei: Acipenseridae) are a group of "living fossil" fishes at a basal position among Actinopteri. They have raised great public interest due to their special evolutionary position, species conservation challenges, as well as their highly-prized eggs (caviar). The sterlet, Acipenser ruthenus, is a relatively small-sized member of sturgeons and has been widely distributing in both Europe and Asia. In this study, we performed whole genome sequencing, de novo assembly and gene annotation of the tarlet to construct its draft genome. Findings: We finally obtained a 1.83-Gb genome assembly (BUSCO completeness of 81.6%) from a total of 316.8-Gb raw reads generated by an Illumina Hiseq 2500 platform. The scaffold N50 and contig N50 values reached 191.06 and 18.88 kb, respectively. The sterlet genome was predicted to be comprised of 42.84% repeated sequences and to contain 22,184 protein-coding genes, of which 21,112 (95.17%) have been functionally annotated with at least one hit in public databases. A genetic phylogeny demonstrated that the sterlet is situated in the basal position among ray-finned fishes and 4dTv analysis estimated that a recent whole genome duplication occurred 21.3 million years ago. Moreover, seven Hox clusters carrying 68 Hox genes were characterized in the sterlet. Phylogeny of HoxA clusters in the sterlet and American paddlefish divided these sturgeons into two groups, confirming the independence of each lineage-specific genome duplication in Acipenseridae and Polyodontidae. Conclusions: This draft genome makes up for the lack of genomic and molecular data of the sterlet and its Hox clusters. It also provides a genetic basis for further investigation of lineage-specific genome duplication and the early evolution of ray-finned fishes.

Optimization of In Vitro Culture Conditions of Sturgeon Germ Cells for Purpose of Surrogate Production.

To expand germ cell populations and provide a consistent supply for transplantation, we established basal culture conditions for sturgeon germ cells and subsequently increased their mitotic activity by eliminating gonad somatic cells, supplementing with growth factor, and replacing fetal bovine serum (FBS). The initial basal culture conditions were Leibovitz's L-15 medium (pH 8.0) supplemented with 5% FBS (p < 0.001) at 21 °C. Proliferation of germ cells was significantly enhanced and maintained for longer periods by elimination of gonad somatic cells and culture under feeder-cell free conditions, with addition of leukemia inhibitory factor and glial-cell-derived neurotrophic factor (p < 0.001). A serum-free culture medium improved germ cell proliferation compared to the L-15 with FBS (p < 0.05). Morphology remained similar to that of fresh germ cells for at least 40 d culture. Germline-specific gene expression analysis revealed no significant changes to germ cells before and after culture. Sterlet Acipenser ruthenus germ cells cultured more than 40 days showed development after transplant into Russian sturgeon Acipenser gueldenstaedtii. Polymerase chain reaction showed 33.3% of recipient gonads to contain sterlet cells after four months. This study developed optimal culture condition for sturgeon germ cells. Germ cells after 40 d culture developed in recipient gonads. This study provided useful information for culture of sturgeon germ cells.

Feedback regulation of 17ß-estradiol on two kisspeptin genes in the Dabry's sturgeon (Acipenser dabryanus).

In tetrapods, kisspeptins are a group of peptides that play essential roles in the regulation of the Gonadotropin-releasing hormone secretion, and may participate in the feedback regulation of sex steroids as well. In this study, two kiss paralogs, designated as dskiss1 and dskiss2 were identified in Acipenser dabryanus. The full-length cDNA sequences of dskiss1 and dskiss2 are 1265 and 744 base pairs (bp), encoding 130 and 146 amino acids, respectively. Multiple sequence alignment indicated that both Kiss1 and Kiss2 decapeptides were highly conserved among vertebrates. Besides, Kiss1 of Dabry's sturgeon shared closer evolutionary relationship with the holostean species spotted gar (Lepisosteus oculatus), while Kiss2 of Acipenser dabryanus was conservatively grouped with the early sarcopterygian coelacanth (Latimeria chalumnae) in the phylogenetic analysis. Tissue distribution analysis showed that dskiss1 transcribed exclusively in the brain, whereas dskiss2 exhibited wider tissue distribution including brain, testis and ovary. Furthermore, male Dabry's sturgeons were intraperitoneally injected with 17ß-estradiol (E2) and the effect of E2 on hypothalamus kiss and its receptors kissr mRNA levels was evaluated by relative real-time PCR. The transcription levels of dskiss2 and dskissr1 were significantly increased by E2 injection (P < .05). However, the mRNA levels of dskiss1 and dskissr2 were not changed in E2-treated group compared to the control group. These results indicate that E2 exerts positive feedback effects through dskiss2/dskissr1 in male Dabry's sturgeon.

Characterization and expression analysis of g- and c-type lysozymes in Dabry's sturgeon (Acipenser dabryanus).

Dabry's sturgeon (Acipenser dabryanus) is mainly distributed in the upper Yangtze River. Although extensively farmed, little information is available on its innate immune system. In this study, we conducted de novo transcriptome assembly of the head kidney to create a comprehensive dataset for A. dabryanus. A total of 51,324,686 high quality reads were obtained from head kidney cDNA library by the Illumina sequencing platform and 131,261 unigenes were determined to contain complete ORFs. The complete coding sequences of g- and c-type lysozymes were identified from unigenes, and designated as ADLysG and ADLysC. Aeromonas hydrophila infection of Dabry's sturgeon caused a significant increase (P < 0.05) in blood for both lysozyme types, confirming their active defensive role against bacterial infections. This research provides the first characterization of these enzymes in an ancestral chondrostean. These data suggest that ADLysG and ADLysC have the potential for immune defense system against bacterial infection.

Molecular characterization, tissue distribution, localization and mRNA expression of the bucky ball gene in the Dabry's sturgeon (Acipenser dabryanus) during oogenesis.

In many organisms, germ cells are specified during embryogenesis by the inheritance of maternally deposited RNAs and proteins termed germ plasm. In vertebrates, the bucky ball (buc) gene plays an essential role in the germ plasm aggregation. In this study, the full-length cDNA of buc homologue in Dabry's sturgeon, Adbuc, was isolated and characterized. Multiple sequence alignments showed that the BUVE domain of Buc was highly conserved in vertebrates, despite exhibiting low identities with each other across the whole protein. By quantitative real-time PCR analysis, we found that Adbuc RNAs were only detected in the gonad with a high level in the ovary and a very low level in the testis. During embryogenesis, these RNAs were highly expressed from the unfertilized eggs to blastula, declined dramatically from the gastrula stage, and hardly found after the neurula stage. Moreover, with the development of ovary, the expression level of Adbuc was increasing. By in situ hybridization, the signal of Adbuc was not found in the oogonia, increased slightly in the stage I oocytes, and extremely strong in the stage II oocytes, suggesting that the signal became much stronger with increasing size of oocytes. Additionally, Adbuc co-localized with the mitochondrial cloud. Thus, we conclude that Dabry's sturgeon buc gene might also function in germplasm formation.

Variability in the protein profiles in spermatozoa of two sturgeon species.

Conventional sperm analysis (i.e., motility and fertility) has been used to evaluate sperm quality. Understanding the quality of sperm on the molecular level in the sturgeons, Acipenser baerii and A. schrenckii, is essential for the improvement of the conservation of genetic resources and farming performance. In this study, we used the iTRAQ proteomics approach to perform proteomic profiling of spermatozoa associated with sperm quality in sturgeons (Data are available via ProteomeXchange with identifier PXD006108). The results showed 291 and 359 differentially expressed proteins in A. baerii and A. schrenckii, respectively, of which 72 were common to both species and all were upregulated in high quality compared with low quality samples. The differentially expressed proteins were mainly categorized into the generation of precursor metabolites and energy and oxidation, and they were localized to the mitochondria. Three distinguishing pathways, Arginine and proline metabolism, Pyruvate metabolism and the Citrate cycle (TCA cycle) were found to play an important role in energy metabolism, and some substrates could be used in the sperm medium for storage and cryopreservation. The quantity levels of two proteins, CKMT1 and LDHB, were verified by western blot analysis. Moreover, other potential biomarkers involved in oxidation reduction, ubiquitin-proteasome-dependent proteolysis, chaperones and binding activity were also discussed. Our study is the first to use the iTRAQ-based proteomics approach to analyse the sturgeon spermatozoa proteome, and the results that we obtained are valuable for the prediction of sperm quality and reproduction management in these threatened species.

Establishment of intraperitoneal germ cell transplantation for critically endangered Chinese sturgeon Acipenser sinensis.

Recent progress in germ cell transplantation techniques in fish has paved the way for the conservation of endangered species. Here, we developed an intraperitoneal germ cell transplantation procedure using Chinese and Dabry's sturgeon as donor and recipient species, respectively. Histological analysis revealed that primordial germ cells migrated on the peritoneal wall at 16 days post-hatch (dph) in Dabry's sturgeon. The genital ridges of Dabry's sturgeon (recipient) first formed at 28 dph, suggesting that for successful colonization of donor germ cells in the recipient gonads, the transplantation should be performed earlier than this age. Sexual dimorphism of gonadal structure was first observed at 78 dph. Gonadal germ cell proliferation was not seen in either sex during this period. Immunohistochemistry using the anti-Vasa antibody found that donor testes from 2-year-old Dabry's sturgeon mainly consisted of single- or paired-type A spermatogonia, while donor ovaries from 11.5-year-old Chinese sturgeon had perinucleolus stage oocytes and clusters of oogonia. Donor cells isolated from Dabry's sturgeon testes or Chinese sturgeon ovary labeled with PKH26 fluorescent dye were transplanted into the peritoneal cavity of the 7- or 8-dph Dabry's sturgeon larvae. More than 90% and 70% of transplanted larvae survived after 2 days post-transplantation (dpt) and 51 dpt, respectively. At 51 dpt, PKH26-labeled cells exhibiting germ cell-specific nuclear morphology and diameter were observed in excised recipient gonads by fluorescent and confocal microscopy. The colonization rate of allogeneic testicular germ cell transplantation (Group 1) was 70%, while that of two batches of xenogeneic ovarian germ cell transplantation (Group 2 and Group 3) were 6.7% and 40%, respectively. The ratio of colonized germ cells to endogenous germ cells was 11.96%, 5.35% and 3.56% for Group 1, Group 2 and Group 3, respectively. Thus, we established a germ cell transplantation technique for the critically endangered Chinese sturgeon using the most closely related species as a recipient and demonstrated the successful preparation of transplantable female germ cells from aged adult Chinese sturgeon.

Identification and sexually dimorphic expression of vasa isoforms in Dabry's sturgeon (Acipenser dabryanus), and functional analysis of vasa 3'-untranslated region.

Germ cells are set aside from somatic cells early in embryogenesis, and are responsible for transmitting genetic information through generations. Vasa is a highly conserved germ cell marker across animal phyla, and widely used to label primordial germ cells. Dabry's sturgeon is a rare and endangered species distributed solely in the Yangtze River basin. Here, seven vasa isoforms, named Advasa1-7, were isolated and characterized in Dabry's sturgeon. RT-PCR and western blot analyses revealed that vasa mRNA and protein were mainly restricted to the testis and ovary, but exhibited sexually dimorphic expression. Cellular and subcellular localization uncovered that Advasa mRNA and protein displayed mitotic and meiotic expression in females, and mainly showed mitotic expression in males; surprisingly, they exhibited both cytoplasmic and nuclear expression in the ovarian germ cells, while showing exclusively cytoplasmic expression in the testicular germ cells. By microinjecting chimeric RNA consisting of the red fluorescent protein coding region and the Advasa 3'-untranslated region into embryos of Dabry's sturgeon, zebrafish and medaka, we demonstrated that it had the ability to visualize primordial germ cells (PGCs) in Dabry's sturgeon and zebrafish but not in medaka. It seemed that the machinery of vasa 3'UTR RNA localization was conserved between Dabry's sturgeon and ostariophysan, while possibly changed during the divergence of euteleosts and ostariophysan. Finally, Dabry's sturgeon PGCs moved on the yolk ball, and migrated toward the genital ridge via mesenchyme. Taken together, these results provide new information for vasa expression pattern and function, and lay a foundation for PGC cryopreservation and conservation of Dabry's sturgeon.

Sequencing and De Novo Assembly of the Gonadal Transcriptome of the Endangered Chinese Sturgeon (Acipenser sinensis).

BACKGROUND: The Chinese sturgeon (Acipenser sinensis) is endangered through anthropogenic activities including over-fishing, damming, shipping, and pollution. Controlled reproduction has been adopted and successfully conducted for conservation. However, little information is available on the reproductive regulation of the species. In this study, we conducted de novo transcriptome assembly of the gonad tissue to create a comprehensive dataset for A. sinensis. RESULTS: The Illumina sequencing platform was adopted to obtain 47,333,701 and 47,229,705 high quality reads from testis and ovary cDNA libraries generated from three-year-old A. sinensis. We identified 86,027 unigenes of which 30,268 were annotated in the NCBI non-redundant protein database and 28,281 were annotated in the Swiss-prot database. Among the annotated unigenes, 26,152 and 7,734 unigenes, respectively, were assigned to gene ontology categories and clusters of orthologous groups. In addition, 12,557 unigenes were mapped to 231 pathways in the Kyoto Encyclopedia of Genes and Genomes Pathway database. A total of 1,896 unigenes, potentially differentially expressed between the two gonad types, were found, with 1,894 predicted to be up-regulated in ovary and only two in testis. Fifty-five potential gametogenesis-related genes were screened in the transcriptome and 34 genes with significant matches were found. Besides, more paralogs of 11 genes in three gene families (sox, apolipoprotein and cyclin) were found in A. sinensis compared to their orthologs in the diploid Danio rerio. In addition, 12,151 putative simple sequence repeats (SSRs) were detected. CONCLUSIONS: This study provides the first de novo transcriptome analysis currently available for A. sinensis. The transcriptomic data represents the fundamental resource for future research on the mechanism of early gametogenesis in sturgeons. The SSRs identified in this work will be valuable for assessment of genetic diversity of wild fish and genealogy management of cultured fish.