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Ellagic acid is a phenolic compound that exhibits both antimutagenic and anticarcinogenic activity in a wide range of assays in vitro and in vivo. It occurs naturally in some foods such as raspberries, strawberries, grapes, and black currants. In this study, a valid and reliable method based on mechanochemical-assisted extraction (MCAE) and macroporous adsorption resin was developed to extract and prepare ellagic acid from Phyllanthus urinaria L. (PUL). The MCAE parameters, acidolysis, and macroporous adsorption resin conditions were investigated. The key MCAE parameters were optimized as follows: the milling time was 5 min, the ball mill speed was 100 rpm, and the ball mill filling rate was 20.9%. Sulfuric acid with a concentration of 0.552 mol/L was applied for the acidolysis with the optimized acidolysis time of 30 min and acidolysis temperature of 40 °C. Additionally, the XDA-8D macroporous resin was chosen for the purification work. Both the static and dynamic adsorption tests were carried out. Under the optimized conditions, the yield of ellagic acid was 10.2 mg/g, and the content was over 97%. This research provided a rapid and efficient method for the preparation of ellagic acid from the cheaply and easily obtained PUL. Meanwhile, it is relatively low-cost work that can provide a technical basis for the comprehensive utilization of PUL.

The reproductive process is usually controlled by the hypothalamic-pituitary-gonad axis in vertebrates, while Kiss/gonadotropin-releasing hormone (GnRH) system in the hypothalamus is required for mammalian reproduction but dispensable for fish reproduction. The regulation of follicle stimulating hormone/luteinizing hormone (LH) expression in fish species is still unknown. Here, we identified miR-200s on chromosome 23 (chr23-miR-200s) as important regulators for female zebrafish reproduction. Knockout of chr23-miR-200s (chr23-miR-200s-KO) resulted in dysregulated expression of luteinizing hormone beta lhb (luteinizing hormone beta) and some hormone genes in the pituitary as revealed by comparative transcriptome profiling, leading to failure of oocyte maturation and ovulation as well as defects in reproductive duct development. Chr23-miR-200s mainly expressed in the pituitary and regulated lhb expression by targeting the transcription repressor wt1a. Injection of human chorionic gonadotropin (hCG) could rescue the defects of oocyte maturation in chr23-miR-200s-KO zebrafish, whereas GnRH or LHRH-A2 could not, suggesting that Chr23-miR-200s regulated lhb expression in a GnRH-independent pathway. It was remarkable that either injection of carp pituitary extraction, or co-injection of hCG with synthetic oxytocin and vasotocin could greatly rescue the defects of both oocyte maturation and ovulation in chr23-miR-200s-KO zebrafish. Altogether, our findings highlight an important function of chr23-miR-200s in controlling oocyte maturation by regulation LH expression, and oxytocin and vasotocin are potentially responsible for the ovulation in fish species.

The sex-determining genes are found to be variable among different fish species. Yellow catfish (Pelteobagrus fulvidraco) is an important aquaculture fish species in China with XX/XY sex-determining type. Recently, YY super-male yellow catfish has been successfully produced by combining hormonal-induced sex reversal method with sex chromosome-linked markers. Here, we identified a novel PDZ domain-containing gene in yellow catfish designated as pfpdz1, in whose intron the sex-linked marker was located. The coding sequence of pfpdz1 in Y chromosome was identical to that in X chromosome except a missense SNP (A/T) that changes an amino acid (E8V) in the N-terminal region. Pfpdz1 displayed male-specific expression during sex differentiation. Overexpression of pfpdz1 using additive transgenesis induces XX ovary to differentiate into testis-like tissue, while the targeted inactivation of pfpdz1 in Y chromosome using CRISPR/Cas9-mediated mutagenesis triggers ovarian differentiation. Furthermore, we demonstrated that pfpdz1 initiates testicular differentiation through upregulating expression of amh, dmrt1 and sox9a1, as well as downregulating expression of cyp19a1, foxl2 and wnt4. Our data provide functional evidence that pfpdz1 is significant for male differentiation and maintenance in yellow catfish.

Signal transducer and activator of transcription 5b (STAT5b) has been identified as a key downstream mediator of growth hormone (GH) signaling in somatic growth of mammalian. However, the corresponding homologue gene of Stat5b is unknown in fish species. In this study, we generated loss-of-function mutants in stat5.1 and stat5.2, two stat5 homologues existing in zebrafish. In stat5.1-deficient zebrafish, a significant reduction of body length and body weight was detected in the embryos/larvae and adults compared with the wild-type control fish, and sexual size dimorphism in adult zebrafish was also eliminated. However, the stat5.2-deficient zebrafish displayed a normal developmental phenotype during all lifespan. Chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) method was adopted to further investigate the potential transcriptional targets of Stat5 protein and cast much light upon the biological function of Stat5. We identified more than 800 genes as transcriptional targets of Stat5 during zebrafish embryogenesis. KEGG analysis indicated that the Stat5 target gene network is predominantly linked to the metabolic pathways, neuroactive ligand-receptor interaction and JAK-STAT signaling pathways. Further validation studies suggested that Stat5.1 protein could directly regulate the expression of gh1, and stat5.1-mutated zebrafish showed a reduction of gh1 mRNA level. In the present study, stat5.1 was revealed as the corresponding homologue gene of Stat5b in fish species. Additionally, we found a novel molecular interaction between Stat5.1/Stat5b and GH, and unraveled a positive feedback loop Stat5.1-GH-Stat5.1 which is necessary for somatic growth and body development in zebrafish.

STAT (Signal Transducers and Activators of Transcription) gene family members have been revealed to be involved in cell growth and differentiation in vertebrates. Despite their physiological importance, their functions are poorly studied at organ and systemic levels. In this study, we performed a genome-wide analysis using data from invertebrates to vertebrates to identify STAT genes and analyze their evolutionary history. Interestingly, the STAT gene family undergoes genome duplications during the evolutionary history with STAT3 homologues firstly appearing in the basal extant vertebrate, sea lamprey, suggesting its possible roles in spine formation. To investigate the functions of stat3 in fish species, TALEN technology was performed to generate mutant zebrafish lines. Stat3 mutant zebrafish showed no obvious defects at early developmental stage but displayed severe lateral and vertical curvature of the spine (scoliosis), spine fracture and the incomplete bone joints with narrower junction between vertebrae at early juvenile stage, as indicated by Alizarin red and Alcian blue staining, radiography and micro-computed tomography (MicroCT) analysis. Transcriptome analysis reveals dramatic alterations in a number of genes involved in immune and infection response, skeletal development and somatic growth, especially downregulated expression of collagen gene family, in the juvenile stat3 mutant zebrafish. Moreover, most of the collagen genes were detected to have abnormal expression pattern during the formation of spine deformities in stat3 mutants. Our data reveal that stat3 is specially expressed in vertebrates and required for normal spine development and immune function in zebrafish.

Sexual size dimorphism is an interesting phenomenon occurred in many fish species. Wildtype zebrafish exhibits a significant sexual dimorphism in body size at the adult stage. Previous studies indicated that sexual size dimorphism was eliminated in stat5b-mutated zebrafish. Herein, the comparative transcriptome analysis was conducted to observe the genes and pathways involved in sexual size dimorphism. The number of male-biased and female-biased genes was much less in the liver of stat5b mutant zebrafish than in wildtype. Gene ontology (GO) enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis indicated that multiple pathways related to metabolism were affected upon loss of stat5b function. qRT-PCR results also validated that sexually dimorphic expression of a set of genes was lost when stat5b was mutated. Furthermore, the weighted correlation network analysis (WGCNA) detected many candidate genes related to the growth traits and stat5b function, such as greb1, lepr, and igf2b. Our data suggest that stat5b should regulate the sexually dimorphic gene expression in zebrafish liver and add in understanding of the molecular mechanisms underlying sexual size dimorphism in fish species.

Besides its well-documented roles in cell proliferation, apoptosis, and carcinogenesis, the function of the p53-microRNA axis has been recently revealed in the reproductive system. Recent studies indicated that miR-200 family members are dysregulated in nonobstructive azoospermia patients, whereas their functions remain poorly documented. The aim of this study was to investigate the function of the miR-200 family on zebrafish testis development and sperm activity. There was no substantial difference in testis morphology and histology between wild-type (WT) and knockout zebrafish with deletion of miR-200 cluster on chromosome 6 (chr6-miR-200-KO) or on chromosome 23 (chr23-miR-200-KO). Interestingly, compared with WT zebrafish, the chr6-miR-200-KO zebrafish had no difference on sperm motility, whereas chr23-miR-200-KO zebrafish showed significantly improved sperm motility. Consistently, ectopic expression of miR-429a, miR-200a, and miR-200b, which are located in the miR-200 cluster on chromosome 23, significantly reduced motility traits of sperm. Several sperm motility-related genes, such as amh, wt1a, and srd5a2b have been confirmed as direct targets of miR-200s on chr23. 17α-ethynylestradiol (EE2) exposure resulted in upregulated expression of p53 and miR-429a in testis and impairment of sperm motility. Strikingly, in p53 mutant zebrafish testis, the expression levels of miR-200s on chr23 were significantly reduced and accompanied by a stimulation of sperm motility. Moreover, the upregulation of miR-429a associated with EE2 treatment was abolished in testis with p53 mutation. And the impairment of sperm activity by EE2 treatment was also eliminated when p53 was mutated. Together, our results reveal that miR-200 cluster on chromosome 23 controls sperm motility in a p53-dependent manner.

Increasing attention has been focused on the role of microRNAs in post-transcription regulation during spermatogenesis. Recently, the miR-34 family has been shown to be involved in the spermatogenesis, but the clear function of the miR-34 family in spermatogenesis is still obscure. Here we analyzed the function of miR-34a, a member of the miR-34 family, during spermatogenesis using miR-34a knockout zebrafish generated by the clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) system. miR-34a knockout zebrafish showed no obvious defects on testis morphology and sperm quantity. However, we found a significant increase in progressive sperm motility that is one of the pivotal factors influencing in vitro fertilization rates, in the knockout zebrafish. Moreover, breeding experiments showed that, when miR-34a-knockout male zebrafish mated with the wide-type females, they had a higher fertilization rate than did the wide-type males. Glycogen synthase kinase-3a (gsk3a), a potential sperm motility regulatory gene was predicted to be targeted by miR-34a, which was further supported by luciferase reporter assays, since a significant decrease of luciferase activity was detected upon ectopic overexpression of miR-34a. Our findings suggest that miR-34a downregulates gsk3a by targeting its 3′ untranslated region, and miR-34a/gsk3a interaction modulates sperm motility in zebrafish. This study will help in understanding in the role of the miR-34 family during spermatogenesis and will set paths for further studies.

Sexual size dimorphism is the consequence of differential expression of sex-biased genes related to feeding and growth. Leptin is known to regulate energy balance by regulating food intake. In order to investigate the molecular mechanism of sexual size dimorphism in yellow catfish (Pelteobagrus fulvidraco), the expression of leptin (lep) and its functional receptor (lepr) were detected during larval development. Both lep and lepr have lower expression in males than in females during 1–4 weeks post hatching. 17a-Methyltestosterone (MT) treatment resulted in decreased expression of lep and lepr in both male and female larval fish. Interestingly, the mRNA levels of lep and lepr in juvenile male were significantly decreased compared with juvenile female during short-term fasting periods. Lep was predicted to be a potential target of miR-200a and miR-200b that had an opposite expression pattern to lep in male and female larvas. The results of luciferase reporter assay suggested that lep is a target of miR-200a/-200b. Subsequently, male hormone and fasting treatment have opposite effects on the expression of miR-200a/-200b and lep between males and females. In summary, our results suggest that sexual size dimorphism in fish species is probably caused by the sexually dimorphic expression of leptin, which could be negatively regulated by miR-200a/-200b.

Background 21-deoxycortisol (21-DF) is the key intermediate to manufacture pharmaceutical glucocorticoids. Recently, a Japan patent has realized 21-DF production via biotransformation of 17-hydroxyprogesterone (17-OHP) by purified steroid 11β-hydroxylase CYP11B1. Due to the less costs on enzyme isolation, purification and stabilization as well as cofactors supply, whole-cell should be preferentially employed as the biocatalyst over purified enzymes. No reports as so far have demonstrated a whole-cell system to produce 21-DF. Therefore, this study aimed to establish a whole-cell biocatalyst to achieve 21-DF transformation with high catalytic activity and product specificity. Results In this study, Escherichia coli MG1655(DE3), which exhibited the highest substrate transportation rate among other tested chassises, was employed as the host cell to construct our biocatalyst by co-expressing heterologous CYP11B1 together with bovine adrenodoxin and adrenodoxin reductase. Through screening CYP11B1s (with mutagenesis at N-terminus) from nine sources, Homo sapiens CYP11B1 mutant (G25R/G46R/L52 M) achieved the highest 21-DF transformation rate at 10.6 mg/L/h. Furthermore, an optimal substrate concentration of 2.4 g/L and a corresponding transformation rate of 16.2 mg/L/h were obtained by screening substrate concentrations. To be noted, based on structural analysis of the enzyme-substrate complex, two types of site-directed mutations were designed to adjust the relative position between the catalytic active site heme and the substrate. Accordingly, 1.96-fold enhancement on 21-DF transformation rate (to 47.9 mg/L/h) and 2.78-fold improvement on product/by-product ratio (from 0.36 to 1.36) were achieved by the combined mutagenesis of F381A/L382S/I488L. Eventually, after 38-h biotransformation in shake-flask, the production of 21-DF reached to 1.42 g/L with a yield of 52.7%, which is the highest 21-DF production as known. Conclusions Heterologous CYP11B1 was manipulated to construct E. coli biocatalyst converting 17-OHP to 21-DF. Through the strategies in terms of (1) screening enzymes (with N-terminal mutagenesis) sources, (2) optimizing substrate concentration, and most importantly (3) rational design novel mutants aided by structural analysis, the 21-DF transformation rate was stepwise improved by 19.5-fold along with 4.67-fold increase on the product/byproduct ratio. Eventually, the highest 21-DF reported production was achieved in shake-flask after 38-h biotransformation. This study highlighted above described methods to obtain a high efficient and specific biocatalyst for the desired biotransformation. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0720-y) contains supplementary material, which is available to authorized users.

Dmrt2b (doublesex and mab-3 related transcription factor 2b) has been revealed to be involved in zebrafish slow muscle development. However, the function of dmrt2a, a paralogue gene of dmrt2b, remains unclear during zebrafish muscle development. Here, we demonstrated that knockdown of dmrt2a resulted in severe developmental defects, and caused downregulation of fast muscle marker myhz-2 and upregulation of slow muscle marker myhz-5, respectively. It is known that microRNAs (miRNAs) control many biological events including muscle development. Dmrt2a was predicted to be a target gene of miR-203, which was further verified by luciferase reporter assay, since miR-203a was found to directly reduce the expression of dmrt2a by binding to the seed sequence of its 3'UTR. After miR-203a injection into zebrafish embryos, the expression of dmrt2a was significantly inhibited. Similar to the effect of dmrt2a knockdown, miR-203a overexpression led to downregulation of myhz-2 and upregulation of myhz-5. Our studies indicated that miR-203a directly regulated dmrt2a expression to control fast and slow muscle differentiation, while overexpression of miR-203a or knockdown of dmrt2a will impair fast muscle development and promote slow muscle development.

In vertebrates, growth hormone/insulin-like growth factor (GH/IGF) axis signaling plays a critical role in regulating somatic growth. Understanding the direct upstream regulators of GH/IGF axis remains a major challenge. Our studies of the zebrafish reveal that the conserved miR-200 family members are critical regulators of embryo size by targeting several GH/IGF axis genes, including GH, GHRa, GHRb and IGF2a. Overexpression of miR-200s led to cell cycle arrest in the G1 phase and induced apoptotic responses during embryo development, thereby inhibiting somatic growth of zebrafish embryos. Intriguingly, GH induced expression of both p53 and miR-200s and miR-200s is a potential p53 transcriptional target, thus forming a negative feedback loop. Significantly, the up-regulation of miR-200s associated with GH activation is abolished in embryos with p53 mutation. By integrating these studies, we conclude that p53/miR-200 and GH/IGF signaling pathway form a negative regulatory loop to control embryo size, that provide critical insights into the long-standing puzzle of how body growth is determined during early development of teleosts.

YY super-males have rarely been detected in nature and only been artificially created in some fish species including tilapia and yellow catfish (Pelteobagrusfulvidraco), which provides a promising model for testis development and spermatogenesis. In our previous study, significant differences in morphology and miRNA expression were detected between XY and YY testis of yellow catfish. Here, solexa sequencing technology was further performed to compare mRNA expression between XY and YY testis. Compared with unigenes expressed in XY testis, 1146 and 1235 unigenes have significantly higher and lower expression in YY testis, respectively. 605 differentially expressed unigenes were annotated to 1604 GO terms with 319 and 286 genes having relative higher expression in XY and YY testis. KEGG analysis suggested different levels of PI3K-AKT and G protein-coupled receptor (GPCR) signaling pathways between XY and YY testis. Down-regulation of miR-141/429 in YY testis was speculated to promote testis development and maturation, and several factors in PI3K-AKT and GPCR signaling pathways were found as predicted targets of miR-141/429, several of which were confirmed by dual-luciferase reporter assays. Our study provides a comparative transcriptome analysis between XY and YY testis, and reveals interactions between miRNAs and their target genes that are possibly involved in regulating testis development and spermatogenesis.

Yellow catfish ( Pelteobagrus fulvidraco ) is an important freshwater fish species in China. In particular, an all-male population has been commercially produced for the males grow faster than females. However, the molecular mechanisms underlying sexual dimorphism of body size and sex differentiation are still unclear in yellow catfish. This study attempts to characterize and analyze the expression of Cytochrome P450 ( CYP ) family members that have been shown to play an important role in sex differentiation and metabolism in teleosts. A total of 25 CYP genes were identified from our transcriptomes by 454 pyrosequencing and Solexa sequencing, including 17 genes with complete open reading frame (ORF). Phylogenetic analyses were conducted to compare these genes with their counterparts from other teleosts. In the tissues of hypothalamic–pituitary–gonad (HPG) axis, most of the genes were expressed at uniform level in both sexes. However, multiple CYP genes displayed sexual dimorphic expression, such as cyp2AD , cyp4b , cyp8a , cyp11b2 , cyp17a and cyp27a expressed at higher level in testis than in ovary, whereas cyp2g , cyp7a , cyp8b , cyp19a1a and cyp26a expressed at higher level in ovary than in testis. The expression response of six CYP genes in ovary was also assessed after 17α-methyltestosterone (MT) treatment. Testis-biased expressed cyp11b2 and cyp17a were significantly up-regulated, while cyp11a and cyp19a1a were reduced in ovary after MT treatment. Our work is helpful for understanding molecular evolution of CYP genes in vertebrates and the mechanism of sexual dimorphism in teleosts.

Recently, YY super-male yellow catfish had been created by hormonal-induced sex reversal and sex-linked markers, which provides a promising research model for fish sex differentiation and gonad development, especially for testis development. MicroRNAs (miRNAs) have been revealed to play crucial roles in the gene regulation and gonad development in vertebrates. In this study, three small RNA libraries constructed from gonad tissues of XX female, XY male and YY super-male yellow catfish were sequenced. The sequencing data generated a total of 384 conserved miRNAs and 113 potential novel miRNAs, among which 23, 30 and 14 miRNAs were specifically detected in XX ovary, XY testis, and YY testis, respectively. We observed relative lower expression of several miR-200 family members, including miR-141 and miR-429 in YY testis compared with XY testis. Histological analysis indicated a higher degree of testis maturity in YY super-males compared with XY males, as shown by larger spermatogenic cyst, more spermatids and fewer spermatocytes in the spermatogenic cyst. Moreover, five miR-200 family members were significantly up-regulated in testis when treated by 17α-ethinylestradiol (EE2), high dose of which will impair testis development and cell proliferation. The down-regulation of miR-141 and 429 coincides with the progression of testis development in both yellow catfish and human. At last, the expression pattern of nine arbitrarily selected miRNAs detected by quantitative RT-PCR was consistent with the Solexa sequencing results. Our study provides a comprehensive miRNA transcriptome analysis for gonad of yellow catfish with different sex genotypes, and identifies a number of sex-biased miRNAs, some of that are potentially involved in testis development and spermatogenesis.