Castration reshapes the liver by altering fatty acid composition and metabolism in male mice.

Castration promotes subcutaneous fat deposition that may be associated with metabolic adaptations in the liver. However, fatty acid composition, abundance, and metabolic characteristics of the liver after castration are not fully understood. Our results showed that surgical castration significantly reduced water and food intake, reduced liver weight, and induced liver inflammation in mice. Transcriptome analyses revealed that castration enhanced fatty acid metabolism, particularly that of arachidonic and linoleic acids metabolism. Gas chromatography-mass spectrometry analysis revealed that castration altered the composition and relative abundance of fatty acids in the liver. The relative abundances of arachidonic and linoleic acids were significantly decreased in 4-week-old castrated mice. Analysis of fatty acid synthesis- and metabolism-related genes revealed that castration enhanced the transcription of fatty acid synthesis- and oxidation-related genes. Analyzing the level of key enzymes in the ß-oxidation and tricarboxylic acid cycle pathways of fatty acids in mitochondria, we found that castration enhanced the ß-oxidation of fatty acids in mitochondria, and also enhanced the protein level of the rate-limiting enzyme in the tricarboxylic acid cycle pathway, isocitrate dehydrogenase 2. These results comprehensively clarify metabolic changes in liver fatty acids after castration in mice of different ages and provide a reference for understanding castration-induced fat deposition from the perspective of liver fatty acid metabolism in male mice.

Testosterone regulates thymic remodeling by altering metabolic reprogramming in male rats.

The thymus is an energy-consuming organ, and its metabolism changes with atrophy. Testosterone regulates thymus remodeling (atrophy and regeneration). However, the characteristics of the energy metabolism during testosterone-mediated thymic atrophy and regeneration remain unclear. In this study, we demonstrated that testosterone ablation (implemented by immunocastration and surgical castration) induced global metabolic changes in the thymus. Kyoto Encyclopedia of Genes and Genomes pathway enrichment for differential metabolites and metabolite set enrichment analysis for total metabolites revealed that testosterone ablation affected thymic glycolysis, glutamate metabolism, and fatty acid ß-oxidation. Testosterone ablation-induced thymic regeneration was accompanied by attenuated glycolysis and glutamate metabolism and changed fatty acid composition and content. Testosterone supplementation in immunocastrated and surgically castrated rats enhanced glutaminolysis, reduced the level of unsaturated fatty acids, enhanced the ß-oxidation of unsaturated fatty acids in the mitochondria, boosted the tricarboxylic acid (TCA) cycle, and accelerated thymic atrophy. Overall, these results imply that metabolic reprogramming is directly related to thymic remodeling.

UNC93B1 facilitates TLR18-mediated NF-κB signal activation in Schizothorax prenanti.

Toll-like receptor 18 (TLR18), a non-mammalian TLR, has been believed to play an important role in anti-bacterial immunity of teleost fishes. UNC93B1 is a classical molecular chaperone that mediates TLRs transport from endoplasmic reticulum to the located membrane. However, TLR18-mediated signal transduction mechanism and the regulatory effect of UNC93B1 to TLR18 are still unclear in teleost fishes. In this study, the coding sequences of TLR18 and UNC93B1 were cloned from Schizothorax prenanti, named spTLR18 and spUNC93B1, respectively. The spTLR18 and spUNC93B1 are 2583 bp and 1878 bp in length, encode 860 and 625 amino acids, respectively. The spTLR18 widely expressed in various tissues with the highest expression level in liver. After stimulation of Aeromonas hydrophila, lipopolysaccharide (LPS) and Poly(I:C), the expression levels of spTLR18 were significantly increased in spleen and head kidney. The spTLR18 located in the cell membrane, while spUNC93B1 located in the cytoplasm. Luciferase and overexpression analysis showed that spTLR18 activated NF-κB and type I IFN signal pathways, and spTLR18-mediated NF-κB activation might depend on the adaptor molecule MyD88. Besides, spUNC93B1 positively regulates spTLR18-mediated NF-κB signal. Our study first uncovers TLR18-UNC93B1-mediated signal transduction mechanism, which contributes to the understanding of TLR signaling pathway in teleost fishes.

PNX14 but not PNX20 as a novel regulator of preadipocyte differentiation via activating Epac-ERK signaling pathway in Gallus gallus.

Small integral membrane protein 20 (SMIM20) could generate two main peptides, PNX14 and PNX20, which participate in multiple biological roles such as reproduction, inflammation and energy metabolism in mammals. However, little is known about their physiological functions in non-mammalian vertebrates. Using chicken (c-) as an animal model, we found cSMIM20 was moderately expressed in adipose tissues, and its expression was gradually increased during the differentiation of chicken preadipocytes, suggesting that it may play an important role in chicken adipogenesis. Further research showed cPNX14 could facilitate the differentiation of chicken preadipocytes into mature adipocytes by enhancing expression of adipogenic genes including PPARγ, CEBPα and FABP4, and promoting the formation of lipid droplets. This pro-adipogenic effect of cPNX14 was completely attenuated by Epac-specific and ERK inhibitor. Interestingly, cPNX20 failed to regulate the adipogenic genes and lipid droplet content. Collectively, our findings reveal that cPNX14 but not cPNX20 can serve as a novel adipogenesis mediator by activating the Epac-ERK signaling pathway in chickens.

Mechanic Insight into the Distinct and Common Roles of Ovariectomy Versus Adrenalectomy on Adipose Tissue Remodeling in Female Mice.

Dysfunctions of the ovaries and adrenal glands are both evidenced to cause aberrant adipose tissue (AT) remodeling and resultant metabolic disorders, but their distinct and common roles are poorly understood. In this study, through biochemical, histological and RNA-seq analyses, we comprehensively explored the mechanisms underpinning subcutaneous (SAT) and visceral adipose tissue (VAT) remodeling, in response to ovariectomy (OVX) versus adrenalectomy (ADX) in female mice. OVX promoted adipocyte differentiation and fat accumulation in both SAT and VAT, by potentiating the Pparg signaling, while ADX universally prevented the cell proliferation and extracellular matrix organization in both SAT and VAT, likely by inactivating the Nr3c1 signaling, thus causing lipoatrophy in females. ADX, but not OVX, exerted great effects on the intrinsic difference between SAT and VAT. Specifically, ADX reversed a large cluster of genes differentially expressed between SAT and VAT, by activating 12 key transcription factors, and thereby caused senescent cell accumulation, massive B cell infiltration and the development of selective inflammatory response in SAT. Commonly, both OVX and ADX enhance circadian rhythmicity in VAT, and impair cell proliferation, neurogenesis, tissue morphogenesis, as well as extracellular matrix organization in SAT, thus causing dysfunction of adipose tissues and concomitant metabolic disorders.

Efficacy of Immunization against a Novel Synthetic 13-Amino Acid Betaglycan-Binding Peptide Sequence of Inhibin α Subunit on Promoting Fertility in Female Rats.

Inhibins suppress the FSH production in pituitary gonadotrope cells by robustly antagonizing activin signaling by competitively binding to activin type II receptors (ACTR II). The binding of inhibin A to ACTR II requires the presence of its co-receptor, namely, betaglycan. In humans, the critical binding site for betaglycan to inhibin A was identified on the inhibin α subunit. Through conservation analysis, we found that a core 13-amino-acid peptide sequence within the betaglycan-binding epitope on human inhibin α subunit is highly conserved across species. Based on the tandem sequence of such a conserved 13-amino-acid betaglycan-binding epitope (INHα13AA-T), we developed a novel inhibin vaccine and tested its efficacy in promoting female fertility using the female rat as a model. Compared with placebo-immunized controls, INHα13AA-T immunization induced a marked (p < 0.05) antibody generation, enhanced (p < 0.05) ovarian follicle development, and increased ovulation rate and litter sizes. Mechanistically, INHα13AA-T immunization promoted (p < 0.05) pituitary Fshb transcription and increased (p < 0.05) serum FSH and 17ß-estradiol concentrations. In summary, active immunization against INHα13AA-T potently increased FSH levels, ovarian follicle development, ovulation rate and litter sizes, thus causing super-fertility in females. Therefore, immunization against INHα13AA is a promising alternative to the conventional approach of multiple ovulation and super-fertility in mammals.

Spexin2 Is a Novel Food Regulator in Gallus gallus.

Spexin2 (SPX2), a paralog of SPX1, is a newly identified gene in non-mammalian vertebrates. Limited studies in fish have evidenced its important role in food intake and energy balance modulation. However, little is known about its biological functions in birds. Using the chicken (c-) as a model, we cloned the full-length cDNA of SPX2 by using RACE-PCR. It is 1189 base pair (bp) in length and predicted to generate a protein of 75 amino acids that contains a 14 amino acids mature peptide. Tissue distribution analysis showed that cSPX2 transcripts were detected in a wide array of tissues, with abundant expression in the pituitary, testis, and adrenal gland. cSPX2 was also observed to be ubiquitously expressed in chicken brain regions, with the highest expression in the hypothalamus. Its expression was significantly upregulated in the hypothalamus after 24 or 36 h of food deprivation, and the feeding behavior of chicks was obviously suppressed after peripheral injection with cSPX2. Mechanistically, further studies evidenced that cSPX2 acts as a satiety factor via upregulating cocaine and amphetamine regulated transcript (CART) and downregulating agouti-related neuropeptide (AGRP) in hypothalamus. Using a pGL4-SRE-luciferase reporter system, cSPX2 was demonstrated to effectively activate a chicken galanin II type receptor (cGALR2), a cGALR2-like receptor (cGALR2L), and a galanin III type receptor (cGALR3), with the highest binding affinity for cGALR2L. Collectively, we firstly identified that cSPX2 serves as a novel appetite monitor in chicken. Our findings will help clarify the physiological functions of SPX2 in birds as well as its functional evolution in vertebrates.

RNA-seq coupling two different methods of castration reveals new insights into androgen deficiency-caused degeneration of submaxillary gland in male Sprague Dawley rats.

BACKGROUND: Salivary gland (SMG) degeneration and dysfunction are common symptoms that occur after sex hormone deprivation, but the underlying mechanisms remain largely unknown. Additionally, immunocastration, which causes drop of sex hormones, has been developed as an alternative to surgical castration, however whether it exerts similar effects as surgical castration on the salivary glands is unknown. Through histological and RNA-seq analysis, we assessed changes in morphology and transcriptome of SMG in response to immunocastration (IM) versus surgical castration (bilateral orchiectomy, ORC). RESULTS: Compared to entire males (EM), ORC caused severe degeneration of SMG in rats, as evidenced by both decreased (P < 0.01) SMG weight and organ index, and by decreased (P < 0.01) quantity of SMG acini and ducts. IM had minimal effects (P > 0.05) on SMG weight and organ index, but it still caused degeneration (P < 0.05) of the acini and ducts. Even though, the quantity of both SMG acini and ducts was much higher (P < 0.001) in IM than in ORC. Functional enrichment analysis of the common regulated genes by ORC/IM revealed disrupted epithelial cell development, angiogenesis, anatomical structure morphogenesis and enhanced cell death are associated with SMG degeneration in deprivation of androgens. Integrated data analysis shown that there existed a selective hyperfunction of SMG ribosome and mitochondrion in ORC but not in IM, which might be associated with more severe degeneration of SMG in ORC than in IM. CONCLUSIONS: Our findings suggested that both surgical castration and immunocastration caused SMG degeneration by disrupting epithelial cell development, angiogenesis, anatomical structure morphogenesis and enhancing cell death. But, surgical castration selectively induced hyperfunction of SMG ribosome and mitochondrion, thus causing more severe degeneration of SMG than immunocastration.

Transcriptomic analyses of the HPG axis-related tissues reveals potential candidate genes and regulatory pathways associated with egg production in ducks.

BACKGROUND: Egg production is one of the most important economic traits in the poultry industry. The hypothalamic-pituitary-gonadal (HPG) axis plays an essential role in regulating reproductive activities. However, the key genes and regulatory pathways within the HPG axis dominating egg production performance remain largely unknown in ducks. RESULTS: In this study, we compared the transcriptomic profiles of the HPG-related tissues between ducks with high egg production (HEP) and low egg production (LEP) to reveal candidate genes and regulatory pathways dominating egg production. We identified 543, 759, 670, and 181 differentially expressed genes (DEGs) in the hypothalamus, pituitary, ovary stroma, and F5 follicle membrane, respectively. Gene Ontology (GO) analysis revealed that DEGs from four HPG axis-related tissues were enriched in the "cellular component" category. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the neuroactive ligand-receptor interaction pathway was significantly enriched based on DEGs commonly identified in all four HPG axis-related tissues. Gene expression profiles and Protein-Protein Interaction (PPI) network were performed to show the regulatory relationships of the DEGs identified. Five DEGs encoding secreted proteins in the hypothalamus and pituitary have interaction with DEGs encoding targeted proteins in the ovary stroma and F5 follicle membrane, implying that they were these DEGs might play similar roles in the regulation of egg production. CONCLUSIONS: Our results revealed that neuroactive ligand-receptor interaction pathway and five key genes(VEGFC, SPARC, BMP2, THBS1, and ADAMTS15) were identified as the key signaling pathways and candidate genes within the HPG axis responsible for different egg production performance between HEP and LEP. This is the first study comparing the transcriptomic profiles of all HPG axis-related tissues in HEP and LEP using RNA-seq in ducks to the best of our knowledge. These data are helpful to enrich our understanding of the classical HPG axis regulating the egg production performance and identify candidate genes that can be used for genetic selection in ducks.

Identification and functional characteristics of two TLR5 subtypes in S. grahami.

TLR5, as a member of Toll-like receptors (TLRs) family in mammals, is responsible for recognizing bacterial flagellin and initiating innate immunity, but its function is still unclear in fish species. In this study, two family members of TLR5 were cloned and identified from Sinocyclocheilus grahami (S. grahami), named sgTLR5a and sgTLR5b. The length of coding sequence of sgTLR5a and sgTLR5b is 2,622 bp and 2,658 bp, encoding 873 and 885 amino acids, respectively. Molecular phylogenetic analysis indicates that sgTLR5a and sgTLR5b have the closest genetic relationship with TLR5M (membrane-type) of Cyprinus carpio and Schizothorax prenanti, respectively. sgTLR5a and sgTLR5b were widely expressed in various tested tissues, of which the expression levels were the highest in skin tissue. After stimulations of Aeromonas hydrophila (A. hydrophila) and flagellin, the expression levels of sgTLR5a and sgTLR5b in liver, spleen and head kidney tissues were strongly up-regulated, but LPS stimulation only increased the expression of sgTLR5b in these tissues. The luciferase reporter assay displayed that sgTLR5a and sgTLR5b could specifically recognize bacterial flagellin and A. hydrophila and activate the downstream NF-κB signaling pathway in HEK293T cells. Moreover, the overexpression of sgTLR5a and sgTLR5b in EPC cells up-regulated the expression levels of IL-8 and TNF. sgTLR5a and sgTLR5b were observed to locate in the intracellular region by confocal microscope. Interestingly, we found that the NF-κB signaling pathway was positively regulated by co-transfecting sgTLR5a or sgTLR5b with TLR trafficking chaperone sgUNC93B1. In conclusion, our results reveal sgTLR5a and sgTLR5b may play an important role in antibacterial response by activating the NF-κB signaling pathway.

Characterization of prolactin (PRL) and PRL receptor (PRLR) in Chinese soft-shelled turtle: Molecular identification, ligand-receptor interaction and tissue distribution.

Prolactin (PRL) plays crucial roles in many physiological and pathological processes through activating its specific membrane-anchored receptor (PRLR). Although this ligand-receptor pair has been extensively studied in mammals, birds and fishes, researches examining their significance is rather scarce in reptiles. Additionally, the interaction mechanism of PRL-PRLR has abortively understood across vertebrates, since two tandem repeated ligand-binding domains of PRLR have been identified in birds and few reptiles. To lay the foundation to clarify their roles and ligand-receptor interaction in reptiles, using Chinese soft-shelled turtle as model, the cDNAs containing open reading frame of PRL and PRLR were cloned. The cloned PRL consisted of 710 bp and encoded a precursor of 228 amino acid (-aa), while PRLR was 2658 bp in length and predicted to generate a 828-aa precursor. Furthermore, the recombinant PRL protein with high-purity was prepared from Escherichia coli (E. coli) strain Rosetta gamiB (DE3) by using cobalt resin. Using the 5 × STAT5-Luciferase reporter system, we found PRLR and PRLR-M2 (the PRLR-mutant lacking membrane-distal ligand-binding domain) could be dose-dependently activated by recombinant PRL, thereby triggering the intracellular JAK2-STAT5 signaling cascade, suggesting PRL-PRLR is functional in Chinese soft-shelled turtle, and the membrane-proximal ligand-binding domain of PRLR is the critical domain involving in PRL-binding. Quantitative real-time PCR revealed that PRL was predominantly and abundantly expressed in pituitary, while PRLR exhibited ubiquitous expression in all of the tissues examined. Collectively, our data indicate the PRL-PRLR pair may function in reptiles including Chinese soft-shelled turtle, in a way similar to that in birds.

Two Synthetic Peptides Corresponding to the Human Follicle-Stimulating Hormone ß-Subunit Promoted Reproductive Functions in Mice.

A follicle stimulating hormone (FSH) is widely used in the assisted reproduction and a synthetic peptide corresponding to a receptor binding region of the human (h) FSH-ß-(34−37) (TRDL) modulated reproduction. Furthermore, a 13-amino acid sequence corresponding to hFSH-ß-(37−49) (LVYKDPARPKIQK) was recently identified as the receptor binding site. We hypothesized that the synthetic peptides corresponding to hFSH-ß-(37−49) and hFSH-ß-(34−49), created by merging hFSH-ß-(34−37) and hFSH-ß-(37−49), modulate the reproductive functions, with the longer peptide being more biologically active. In male or female prepubertal mice, a single injection of 200 µg/g BW ip of hFSH-ß-(37−49) or hFSH-ß-(34−49) hastened (p < 0.05) puberty, whereas the same treatments given daily for 4 d promoted (p < 0.05) the gonadal steroidogenesis and gamete formation. In addition of either peptide to the in vitro cell cultures, promoted (p < 0.05) the proliferation of primary murine granulosa cells and the estradiol production by upregulating the expression of Ccnd2 and Cyp19a1, respectively. In adult female mice, 200 µg/g BW ip of either peptide during diestrus antagonized the FSH-stimulated estradiol increase and uterine weight gain during proestrus. Furthermore, hFSH-ß-(34−49) was a more potent (p < 0.05) reproductive modulator than hFSH-ß-(37−49), both in vivo and in vitro. We concluded that hFSH-ß-(37−49) and especially hFSH-ß-(34−49), have the potential for reproductive modulation.

Transcriptome-based evaluation and validation of suitable housekeeping gene for quantification real-time PCR under specific experiment condition in teleost fishes.

Quantification real-time PCR (qRT-PCR) is a common method in analysis of gene expression, but the stable reference genes for the normalization analysis have not been appreciated before identifying expression pattern of genes in teleost fishes. In this study, we selected eight candidate reference genes (18S, Actin, EF-1α, 40S, B2M, TUBA, UBCE and GAPDH) basing on transcriptome analysis and the traditional housekeeping genes, and analyzed the stability of the reference genes in spleen, head kidney and head kidney leukocytes (HKL) after pathogen challenge in Schizothorax prenanti (S. prenanti). Three common programs (geNorm, NormFinder and Bestkeeper) were used to evaluate the stability of the candidate reference genes. Two reference genes, Actin and EF-1α presented higher stability, while 18S and GAPDH were the lower stable genes, both in in vitro and in vivo. An important immune gene, toll-like receptor 22a (TLR22a), was selected to validate the stability of the proposed reference genes (Actin and EF-1α) across different experiment treatments. The results reveal that Actin and EF-1α are quite suitable reference genes for the normalization analysis. Otherwise, using the most stable gene Actin to validate the reliable of transcriptome data showed the high correlation between the fold change of transcriptome data and qRT-PCR data. In conclusion, our study not only acquired the suitable reference gene for the qRT-PCR assay under specific experiment condition, but also provided a comprehensive method to evaluate and validate the reference gene based on transcriptome analysis in teleost fishes.

The potential sensing molecules and signal cascades for protecting teleost fishes against lipopolysaccharide.

Lipopolysaccharide (LPS) is a classical pathogen-associated molecular pattern that can trigger strong inflammatory response mainly by TLR4-mediated signaling pathway in mammals, but the molecular mechanism of anti-LPS immunity is unclear in teleost fishes. In this study, we analyzed the gene expression features based on transcriptome analysis in Schizothorax prenanti (S. prenanti), after stimulation with two sources of LPS from Aeromonas hydrophila and Escherichia coli (Ah. LPS and Ecoli. LPS). 921 different expression genes (DEGs) after Ah. LPS stimulation and 975 DEGs after Ecoli.LPS stimulation were acquired, but only 706 and 750 DEGs were successfully annotated into the databases, respectively. Both of two groups of DGEs were significantly enriched into immune-related pathways by KEGG enrichment analysis, such as "Toll-like receptor signaling pathway", "Cytokine-cytokine receptor interaction" and "JAK-STAT signaling pathway". The annotated DEGs from Ah. LPS and Ecoli. LPS stimulation shared 470 DEGs, including 88 immune-related DEGs (IRGs) identified mainly by KEGG enrichment to immune-related signaling pathways. Among the shared IRGs, four pattern-recognition genes (TLR5, TLR25, PTX3 and C1q) were induced with high expression foldchange, and IFN-γ and relative genes also showed higher expression levels than control. Meanwhile, inflammatory signals were highlighted by upregulating the expression of inflammatory cytokines (IL-1ß, IL-10 and IL-8). Moreover, some non-shared IRGs (including TLR2 and TLR4) were identified, suggesting that different sources of LPS own different potentials for the induction of immune gene expression. In conclusion, TLR5, TLR25, PTX3 and C1q may function as the sensing molecules to catch the invasion signal of LPS. The anti-LPS immune response may be involved into TLR25/TLR5-mediated inflammatory signals that regulate subsequently the activation of PTX3/C1q-modulated complement pathway upon the induction of PTX3 expression, and the crosstalk between IFN-γ and TLR signaling pathways in teleost fishes. This study will contribute to further explore the molecular mechanism of LPS-induced immunity in teleost fishes.

Circular RNAs as biomarkers and therapeutic targets in environmental chemical exposure-related diseases.

Chemical contamination in the environment is known to cause abnormal circular RNA (circRNA) expression through multiple exposure routes; yet, the underlying molecular mechanisms remain unclear. Non-coding RNAs (ncRNAs), especially circRNAs, play important roles in epigenetic regulation and disease pathogenesis; however, few studies have examined the function of circRNAs in chemical contamination-induced diseases. CircRNAs are covalently closed continuous loops that do not possess 5' and 3' ends, increasing their structural stability and limiting degradation by exoribonucleases. In addition, environmental chemical exposure-related diseases are often accompanied by aberrant expression of specific circRNAs and those circRNAs are often detected in tissues and body fluids. Based on these characteristics, circRNAs may serve as candidate biomarkers for the diagnosis of diseases related to environmental chemical exposure. Here, we review the generation and function of circRNAs, and the possible molecular mechanisms underlying the regulation of environmental chemical exposure-related disorders by circRNAs. This is the first comprehensive review of the relationship between environmental chemical exposure and circRNAs in chemical exposure-induced diseases.

Oral oyster polypeptides protect ovary against d-galactose-induced premature ovarian failure in C57BL/6 mice.

BACKGROUND: Oyster polypeptides have various biofunctions, such as anti-cancer and anti-oxidative stress, but whether it has the protective effects to primary ovarian failure (POF) remains poorly understand. To address this issue, daily gavage of oyster polypeptides was performed to investigate their protective effect, basing on d-galactose-induced POF model in C57BL/6 female mice. RESULTS: Oyster polypeptides restored the irregular estrous cycles and the abnormal serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and progesterone (P) levels as well as the decreased mRNA expression level of Amh that were induced by d-galactose. The follicle development of POF mice was improved by increasing the primordial follicle ratio and decreasing the atretic follicle number after oral administration of oyster polypeptides. Moreover, in the oyster polypeptides treated mice, the total superoxide dismutase (T-SOD) activity was significantly increased, while the malondialdehyde levels were significantly decreased. The mRNA expression levels of stress-related genes (SOD2, SIRT1 and FOXO3a) were remarkably up-regulated after d-galactose induction, but the up-regulation was weakened or disappeared by the gavage of oyster polypeptides. In addition, oyster polypeptides treatment also reduced the apoptosis of the ovarian granulosa cells and down-regulated the mRNA expression levels of apoptosis-related genes (p53 and Bad but not Bcl-2). CONCLUSION: This study reveals that oyster polypeptides may protect ovary against d-galactose-induced POF by their anti-oxidative stress activity to rescue d-galactose-induced ovarian oxidative damage and therefore to prevent ovarian cells apoptosis, thereby tipping the abnormality trigged by POF to get close to the normal levels. © 2019 Society of Chemical Industry.

Transcriptome analysis of spleen reveals the signal transduction of toll-like receptors after Aeromonas hydrophila infection in Schizothorax prenanti.

Schizothorax prenanti (S. prenanti), an important species of economical fish in Southwest China, is susceptible to Aeromonas hydrophila (Ah). To understand the immune response to Ah, the transcriptome profiling of spleen of S. prenanti was analyzed after Ah infection. A total of 6, 213 different expression genes (DEGs) were obtained, including 3, 066 up-regulated DEGs and 3, 147 down-regulated DEGs. These DEGs were annotated by KEGG and GO databases, so that the immune-related DEGs (IRDs) can be identified and classified. Then, the interesting IRDs were screened to build heat map, and the reliability of the transcriptome data was validated by qPCR. In order to clarify the mechanism of signal transduction in the anti-bacterial immunity, the signaling pathway initiated by TLRs was predicted. In this pathway, TLR25 and TLR5 mediate the NF-κB and AP-1 signals via MyD88-dependent pathway. Meanwhile, the type I IFN (IFNα/ß) induced by IRF1 and IRF3/7 may play an important role in the anti-bacterial immunity. In conclusion, this study preliminarily provides insights into the mechanism of signal transduction after Ah infection in S. prenanti, which contributes to exploring the complex anti-bacterial immunity.

Clone, identification and functional character of two toll-like receptor 5 molecules in Schizothorax prenanti.

Mammal Toll-like receptor 5 (TLR5) can directly recognize bacterial flagellin, initiate the inflammatory signaling cascades and trigger body immune system to clear the "non-self" substances. In teleosts, TLR5 has presented more complexes not only in increasing the molecular types, but also in elevating the functional diversity. In this study, we identified two TLR5 family members in Schizothorax prenanti, named as spTLR5-1 and spTLR5-2. The complete coding sequence (CDS) of spTLR5-1 is 2622 bp, encoding 873 amino acids, while the complete CDS of spTLR5-2 is 2640 bp, encoding 879 amino acids. Phylogenetic analysis showed that spTLR5-1 and spTLR5-2 were clustered to the TLR5 of schizothorax richardsonii and Cyprinus carpio respectively. The 3D structure analysis exhibited that the α-helix, ß-sheet, and the ligand binding site of spTLR5-1, spTLR5-2 and human TLR5 have large differences. The spTLR5-1 and spTLR5-2 had extensively expressed in various tissues, including the higher expression in liver, spleen and head kidney. Both the expression levels of spTLR5-1 and spTLR5-2 were significantly up-regulated after Aeromonas hydrophila (A. hydrophila) challenge. And, the downstream genes, such as AP-1, IKK-α, NF-kB, IL-1ß, IL-8 and TNF-α, were also significantly up-regulated after A. hydrophila challenge. Apart from that, the luciferase reporter assay demonstrated that the co-transfection of spTLR5-1 or spTLR5-2 into HEK293T cells showed the significantly increased NF-kB luciferase activity after flagellin stimulation. In conclusion, our results reveal that both two molecular types of fish TLR5 may commonly mediate the recognition of flagellin and the activation of the downstream inflammatory signaling molecules.

Multiple subtypes of TLR22 molecule from Schizothorax prenanti present the functional diversity in ligand recognition and signal activation.

Evolutionary development has increased the diversity of genotypes and the complexity of gene functions in fish. TLR22 has been identified as a teleost-specific gene, but its functions are tremendously different among different fish species. Whether the functional diversity relates to the difference of genotypes remains poorly understand. In this study, we cloned and identified three TLR22 molecules from Schizothorax prenanti (S. prenanti), named as spTLR22-1, spTLR22-2 and spTLR22-3. The full-length coding regions of spTLR22s are 2841 bp, 2805 bp and 2868 bp and coding 946 aa, 934 aa and 955 aa, respectively. All spTLR22s are composed of multiple leucine-rich repeat (LRR) domains, a transmembrane structure and a Toll/IL-1 receptor (TIR) region. The phylogenetic analysis showed that three spTLR22s were close to Cyprinus carpio TLR22-1, TLR22-2 and TLR22-3, respectively. Among the spTLR22s, they presented not close relationship but remained to belong to TLR22 subfamily. All spTLR22s were ubiquitously expressed in all tested tissues, but the expression levels of spTLR22s were dominant in immune-related tissues, such as gill and spleen. The expression levels of spTLR22-1 and spTLR22-3 were significantly increased after treatment with bacteria, LPS and Poly(I:C). However, spTLR22-2 seems like no response to these treatments. The luciferase reporter assay demonstrated that all spTLR22s could activate NF-κB signaling pathway, but only spTLR22-1 and spTLR22-2 could activate IFN-ß signaling pathway. Interestingly, in the ligand recognition analysis, spTLR22-1 and spTLR22-3 but not spTLR22-2 had the recognized potential to Poly(I:C), and all spTLR22s could not recognize LPS. Both spTLR22-1 and spTLR22-3 significantly up-regulated the expression of anti-viral-related genes (Mx, IFN and ISG15) and down-regulated the expression of anti-inflammatory factor IL-10 after the overexpression in carp EPC cell line, but spTLR22-2 failed to impact the expression of these genes. Moreover, we found that all spTLR22s localized to the intracellular region. Taken together, our results reveal that spTLR22-1 and spTLR22-3 but not spTLR22-2 may be involved into the anti-viral immune response via IFN-ß signaling pathway, and all spTLR22s can activate NF-κB signaling pathway but only spTLR22-1 and spTLR22-3 response to the stimulation of bacteria and LPS.

Teleost-specific TLR25 identified from Schizothorax prenanti may recognize bacterial/viral components and activate NF-κB and type I IFNs signaling pathways.

TLR25 is a new member of TLR1 family that is only identified in teleosts, but its function in immune response is still unclear. In current study, the coding sequence (CDS) of TLR25 was cloned from Schizothorax prenanti (named spTLR25), and spTLR25 is 2454 bp in length and coding a protein of 817 aa. The spTLR25 contains a signal peptide, twenty leucine-rich repeat (LRR) domains, a LRR C-terminal (LRRCT) motif, a transmembrane region and a Toll/IL-1 receptor (TIR) domain. Phylogenetic analysis indicates that spTLR25 has the closest relationship with Cyprinus carpio (C. carpio) TLR25-2. The 3D structure of spTLR25 exhibits 5 α-helices and 3 ß-sheets in the TIR domain, and 8 α-helices and 6 ß-sheets in the LRR domains. The spTLR25 is mainly expressed in immune-related tissues and peripheral blood leukocytes (PBL). Furthermore, the expression levels of spTLR25 were upregulated in spleen, head kidney and liver while S. prenanti was challenged with LPS or Aeromonas hydrophila (Ah), and the upregulation was also detected in head kidney leukocytes (HKL) after LPS and Poly (I:C) stimulation. The luciferase reporter assay demonstrated that NF-κB and type I IFNs signaling pathways can be activated by spTLR25, and this process may involve in the cascade amplification of TLR25-MyD88 signaling. In addition, the co-localization analysis showed that spTLR25 localizes to intracellular region. Taken together, our results reveal that teleost-specific TLR25 may be a multifunctional receptor for recognizing both LPS and Poly (I:C) and may activate NF-κB and type I IFNs signaling pathways.