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.

TAC3 regulates GnRH/gonadotropin synthesis in female chickens.

Neurokinin B (NKB), a peptide encoded by the tachykinin 3 (TAC3), is critical for reproduction in all studied species. However, its potential roles in birds are less clear. Using the female chicken (c-) as a model, we showed that cTAC3 is composed of five exons with a full-length cDNA of 787 bp, which was predicted to generate the mature NKB peptide containing 10 amino acids. Using cell-based luciferase reporter assays, we demonstrated that cNKB could effectively and specifically activate tachykinin receptor 3 (TACR3) in HEK293 cells, suggesting its physiological function is likely achieved via activating cTACR3 signaling. Notably, cTAC3 and cTACR3 were predominantly and abundantly expressed in the hypothalamus of hens and meanwhile the mRNA expression of cTAC3 was continuously increased during development, suggesting that NKB-TACR3 may emerge as important components of the neuroendocrine reproductive axis. In support, intraperitoneal injection of cNKB could significantly promote hypothalamic cGnRH-Ι, and pituitary cFSHß and cLHß expression in female chickens. Surprisingly, cTAC3 and cTACR3 were also expressed in the pituitary gland, and cNKB treatment significantly increased cLHß and cFSHß expression in cultured primary pituitary cells, suggesting cNKB can also act directly at the pituitary level to stimulate gonadotropin synthesis. Collectively, our results reveal that cNKB functionally regulate GnRH/gonadotropin synthesis in female chickens.

Active immunization against gonadotropin-releasing hormone affects thymic T cell production, migration, and colonization in male rat lymphoid tissue.

Active immunization against gonadotropin-releasing hormone (GnRH) inhibits animal reproduction and has become a friendly alternative to surgical castration, which has been reported to affect the proportion of thymic T cell subpopulations. The effects of active immunization against GnRH on T cell migration from the thymus to the periphery and T cell distribution in lymphoid tissues remain unclear. Here, we showed that active immunization against GnRH increased thymic size and weight, enlarged the number of thymocytes, and enhanced CD4+ recent thymic emigrants (RTEs) and CD8+ RTEs migration to the blood and spleen. Active immunization against GnRH had no significant effect on naïve CD4+, naïve CD8+, CD4+ memory/activated, or CD8+ memory/activated T cells. In addition, active immunization against GnRH increased the proportion of CD3+ T cells in the spleen and lymph nodes. The percentages of CD3+CD4+ and CD3+CD8+ T cells in the blood, spleen, and lymph nodes were not significantly affected by GnRH immunization. Overall, these results enhance our understanding of thymic T cell production, migration, and colonization in rat lymphoid tissues affected by GnRH immunization.

Polymorphisms Analysis of BMP15, GDF9 and BMPR1B in Tibetan Cashmere Goat (Capra hircus).

The Tibetan cashmere goat is a prolific goat breed in China. In sheep breeds, natural mutations have demonstrated that the transforming growth factor beta (TGF-ß) super family ligands, such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor (BMPR1B), are essential for ovulation and increasing litter size. In this study, 216 female Tibetan cashmere goats were sampled, and candidate genes with fecundity traits were detected via restriction fragment length polymorphism (RFLP) and sequenced. Four polymorphic loci were found in specific amplification fragments of BMP15 and GDF9. Two SNP sites of the BMP15 gene were discovered, namely G732A and C805G. The G732A mutation did not cause the change in amino acids, and the frequencies of each genotype were 0.695 for the GG type, 0.282 for the GA type and 0.023 for the AA type. The C805G mutation caused amino acids to change from glutamine to glutamate. The genotype frequencies were 0.620 for the CC type, 0.320 for the CG type and 0.320 for the CG type. For the GG type 0.060, the G3 and G4 mutations of the GDF9 gene were all homozygous mutations. Two known SNP sites, C719T and G1189A, were detected in the Tibetan cashmere goat GDF9 gene, of which the C719T mutation caused a change of alanine to valine, with a genotype frequency of 0.944 for the CC type and 0.056 for the CT type, whereas no TT type was found. The G1189A mutation caused valine to become isoleucine, and the frequencies of each genotype were 0.579 for the GG type, 0.305 for the GA type and 0.116 for the AA type; G1, B2, B3, B4, FecXH, FecXI, FecXL, G2, G5, G6, G7, G8, FecGE, FecTT and FecB mutations were not found in Tibetan cashmere goats. The results of this study provide a data basis for future studies of BMP15, GDF9 and BMPR1B gene mutations in goats.

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.

Polymorphisms and mRNA Expression Levels of IGF-1, FGF5, and KAP 1.4 in Tibetan Cashmere Goats.

The Tibetan cashmere goat is a precious breed in China and its cashmere is widely used in clothing and textiles. The genes IGF-1, FGF5, and KAP 1.4 have been shown to be crucial regulators of cashmere growth. In this study, we examined mRNA expression levels of these three genes and detected IGF-1, FGF5, and KAP 1.4 SNP loci in the Tibetan cashmere goat. After amplification and sequence alignment of the genes IGF-1, FGF5, and KAP 1.4 among 206 Tibetan cashmere goats, two new SNP loci were detected in gene KAP 1.4, while no SNP loci were found in amplified fragments of genes IGF-1 and FGF5. The expression levels of gene IGF-1 in Baingoin and Nyima counties were significantly higher than in other counties (p < 0.05). Moreover, the expression level of gene FGF5 in Gêrzê was significantly higher than in Rutog. The expression levels of mRNA in KAP 1.4 showed significant variation among seven counties. There were no significant differences in mRNA expression levels of IGF-1, FGF5, and KAP 1.4 in Tibetan cashmere goats when analysed by sex. The gene IGF-1 was slightly up-regulated in one to five-year-old cashmere goats, except in those that were 4 years old. The mRNA expression levels of FGF5 in one and two-year-old cashmere goats was lower compared with those in three to five-year-old cashmere goats. KAP 1.4 was up-regulated across one to five-year-old cashmere goats. In this study, SNP detection and mRNA expression analysis of IGF-1, FGF5, and KAP 1.4 genes was able to add data to genetic evolutionary analysis. Further studies should be carried out in SNPs to detect other fragments in genes IGF-1 and FGF5, as well as signal pathways and gene functions in protein levels of genes IGF-1, FGF5, and KAP 1.4 in the Tibetan cashmere goat.

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.

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.

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.

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.

Corticosterone stage-dependently inhibits progesterone production presumably via impeding the cAMP-StAR cascade in granulosa cells of chicken preovulatory follicles.

Stress can suppress reproduction capacity in either wild or domestic animals, but the exact mechanism behind it, especially in terms of steroidogenesis, remains under-investigated so far. Considering the important roles of progesterone in avian breeding, we investigated the modulation of corticosterone on progesterone production in cultured granulosa cells of chicken follicles at different developmental stages. Using enzyme immunoassays, our study showed that corticosterone could only inhibit progesterone synthesis in granulosa cells from F5-6, F4, and F3 follicles, but not F2 and F1 follicles. Coincidentally, both quantitative real-time PCR and western blotting revealed that corticosterone could downregulate steroidogenic acute regulatory protein (StAR) expression, suggesting the importance of StAR in corticosterone-related actions. Using the dual-luciferase reporter system, we found that corticosterone can potentially enhance, rather than inhibit, the activity of StAR promoter. Of note, combining high-throughput transcriptomic analysis and quantitative real-time PCR, phosphodiesterase 10A (PDE10A), protein kinase cAMP-dependent type II regulatory subunit alpha (PRKAR2A) and cAMP responsive element modulator (CREM) were identified to exhibit the differential expression patterns consistent with cAMP blocking in granulosa cells from F5-6, F4, and F3, but not F2 and F1 follicles. Afterward, the expression profiles of these genes in granulosa cells of distinct developmental-stage follicles were examined by quantitative real-time PCR, in which all of them expressed correspondingly with progesterone levels of granulosa cells during development. Collectively, these findings indicate that corticosterone can stage-dependently inhibit progesterone production in granulosa cells of chicken preovulatory follicles, through impeding cAMP-induced StAR activity presumptively.

Thallium(III) exposure alters diversity and co-occurrence networks of bacterial and fungal communities and intestinal immune response along the digestive tract in mice.

The gut microbiota, which includes fungi and bacteria, plays an important role in maintaining gut health. Our previous studies have shown that monovalent thallium [Tl(I)] exposure is associated with disturbances in intestinal flora. However, research on acute Tl(III) poisoning through drinking water and the related changes in the gut microbiota is insufficient. In this study, we showed that Tl(III) exposure (10 ppm for 2 weeks) reduced the alpha diversity of bacteria in the ileum, colon, and feces of mice, as well as the alpha diversity of fecal fungi. In addition, principal coordinate analysis showed that Tl(III) exposure had little effect on the bacterial and fungal beta diversity. LEfSe analyses revealed that Tl(III) exposure altered the abundance of intestinal bacteria in the digestive tract and feces. Moreover, Tl(III) exposure had little effect on fungal abundance in the ileum, cecum, and colon, but had a considerable effect on fungal abundance in feces. After Tl(III) exposure, the fungal composition was more disrupted in feces than in the intestinal tract, suggesting that feces can serve as a representative of the gut mycobiota in Tl(III) exposure studies. Intra-kingdom network analyses showed that Tl(III) exposure affected the complexity of bacterial-bacterial and fungal-fungal co-occurrence networks along the digestive tract. The bacterial-fungal interkingdom co-occurrence networks exhibited increased complexity after Tl(III) exposure, except for those in the colon. Additionally, Tl(III) exposure altered the intestinal immune response. These results reveal the perturbation in gut bacterial and fungal diversity, abundance, and co-occurrence network complexity, as well as the gut immune response, caused by Tl(III) exposure.

Characterization of spexin (SPX) in chickens: molecular cloning, functional analysis, tissue expression and its involvement in appetite regulation.

Spexin (SPX) is a conservative tetradecapeptide which has been proven to participate in multiple physiological processes, including anxiety, feed intake, and energy metabolism in fish and mammals. However, whether SPX exists and functions in birds remain largely unknown. Using chicken (c-) as a model, the full-length cDNA encoding cSPX precursor was cloned, and it was predicted to generate a mature peptide with 14 amino acids conserved across vertebrates. The pGL4-SRE-luciferase reporter system-based functional analysis demonstrated that cSPX was effective in activating chicken galanin type Ⅱ receptor (cGALR2), cGALR2-like receptor (cGALR2L) and galanin type Ⅲ receptor (cGALR3), thus to stimulate intracellular MAPK/ERK signaling pathway. Quantitative real-time PCR revealed that SPX was widely expressed in chicken tissues, especially abundant in the central nervous system, pituitary, testes, and pancreas. Interestingly, it was noted that chicken hypothalamic SPX mRNA could be up-regulated by 24-h and 36-h fasting, heralding its latent capacity in appetite regulation. In accordance with this speculation, peripheral injection of cSPX was proved to be functional in reducing feed intake of 3-wk-old chicks. Furthermore, we found that cSPX could reduce the expression of AgRP and MCH, with a concurrent rise in CART1 mRNA level in the hypothalamic of chicks. Collectively, our findings not only provide the evidences that SPX can act as a satiety factor by orchestrating the expression of key feeding regulators in the chicken hypothalamus but also help to facilitate a better understanding of its functional evolution across vertebrates.

Evidence for progesterone acting as an inhibitor of stress axis via stimulating pituitary neuropeptide B/W receptor 2 (NPBWR2) expression in chickens.

In vertebrates, the hypothalamus-pituitary-adrenal gland (HPA) axis is the main endocrine pathway regulating the stress response, thus also called the stress axis. It has been well-accepted that the stress axis is tightly controlled by both hypothalamic stimulators and inhibitors [e.g. corticotropin (ACTH)-releasing inhibitory factor (CRIF)]. However, the identity of authentic CRIF remains unclear for decades. Recently, neuropeptide W (NPW) was proved to be the physiological CRIF in chickens. Together with its functional receptor (NPBWR2), they play critical roles in attenuating the activity of the chicken stress axis. Because increasing pieces of evidence suggested that sex steroids could regulate the stress axis, using chicken as a model, we investigated whether the newly identified CRIF and its receptor are under the control of sex steroids in this study. Our results showed that: (1) expression of NPW-NPBWR2 in the hypothalamus-pituitary axis was sexually dimorphic and developmental stage-dependent; (2) progesterone (P4), rather than 17ß-estradiol (E2) and dihydrotestosterone (DHT), could dose- and time-dependently upregulate NPBWR2 expression, which was accompanied with the decrease of ACTH synthesis and secretion, in cultured pituitary cells; (3) intraperitoneal injection of P4 could elevate the mRNA level of pituitary NPBWR2; (4) P4-stimulated NPBWR2 expression was relevant to both nPR-mediated genomic action and mPRs-triggered nongenomic route associated with MEK/ERK, PI3K/AKT cascade, and calcium influx. To our knowledge, our results discover a novel route of sex steroids in modulating the stress axis of chickens, which lays a foundation to reveal the complicated interaction network between reproduction and stress axes in chickens.

Research Note: Integrated transcriptomic and metabolomic analysis reveals potential candidate genes and regulatory pathways associated with egg weight in ducks.

Egg weight is an important indicator of egg phenotypic traits, which directly affects the economic benefits of the poultry industry. In the present research, laying ducks were classified into high egg weight (HEW) and light egg weight (LEW) groups. To reveal the underlying mechanism that may be responsible for the egg weight difference, the integrated analysis of transcriptomes and serum metabolomics was performed between the two groups. The results showed extremely significant differences (P < 0.01) in the total egg weight at 300 d, and average egg weight between the HEW and LEW groups. 733, 591, 82, and 74 differentially expressed genes (DEGs) were identified in the liver, magnum, F1, and F5 (hierarchical follicles) follicle membrane, respectively. The candidate genes were screened further from the perspective of forming an egg. In terms of egg yolk formation, the functional analysis revealed fatty acid metabolism-related pathways account for 36% of the liver's top pathways, including fatty acid biosynthesis, folate biosynthesis, fatty acid metabolism, and glycerol lipid metabolism pathways. FASN gene was identified as the key candidate gene by comprehensive analysis of gene expression and protein-protein interaction (PPI) network. In the follicle membrane, the DEGs were mainly enriched in protein processing in the endoplasmic reticulum, and MAPK signaling pathway, and HSPA2, HSPA8, BAG3 genes were identified as crucial candidate genes. In terms of egg white formation, the functional analysis revealed protein metabolism-related pathways account for 40% of the magnum's top pathways, which includes protein processing in the endoplasmic reticulum pathway. HSP90AA1 and HSPA8 genes were identified as key candidate genes. In addition, the integrated transcriptomic and metabolomic analysis showed that arginine and proline metabolism pathways could contribute to differences in egg weight. Thus, we speculated that the potential candidate genes, regulatory pathways, and metabolic biomarkers mentioned above might be responsible for the egg weight difference. These findings might provide a theoretical basis for improving the egg weight of ducks.

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.

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.

Thallium(I) exposure perturbs the gut microbiota and metabolic profile as well as the regional immune function of C57BL/6 J mice.

Intestinal microbes regulate the development of diseases induced by environmental exposure. Thallium (Tl) is a highly toxic heavy metal, and its toxicity is rarely discussed in relation to gut microbes. Herein, we showed that Tl(I) exposure (10 ppm for 2 weeks) affected the alpha diversity of bacteria in the ileum, colon, and feces, but had little effect on the beta diversity of bacteria through 16S rRNA sequencing. LEfSe analysis revealed that Tl(I) exposure changed the abundance of intestinal microbiota along the digestive tract. Cecum metabolomic detection and analysis showed that Tl(I) exposure altered the abundance and composition of metabolites. In addition, the Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analysis revealed that Tl(I) exposure impaired amino acid, lipid, purine metabolism, and G protein-coupled receptor signalling pathways. A consistency test revealed a strong correlation, and a Pearson's correlation analysis showed an extensive interaction, between microorganisms and metabolites. Analysis of the intestinal immunity revealed that Tl(I) exposure suppressed the immune responses, which also had regional differences. These results identify the perturbation of the intestinal microenvironment by Tl exposure and provide a new explanation for Tl toxicity.

Thallium(I and III) exposure leads to liver damage and disorders of fatty acid metabolism in mice.

Thallium (Tl), a highly toxic and priority pollutant heavy metal, exposure can damage mitochondria and disrupt their function. The liver is the central organ that controls lipid homeostasis and contains a large number of mitochondria. So far, there is no study investigating the effects of Tl exposure on hepatic fatty acid metabolism. Here, we showed that 10 ppm of Tl(I) and Tl(III) exposures for two weeks did not significantly affect the body weight and water/food intake in mice. However, it decreased the ratio of liver/weight and induced hepatic sinus congestion and hepatocyte necrosis. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis revealed Tl accumulation in the liver. Gas chromatography-mass spectrometry (GC-MS) results showed that Tl(I) exposure significantly increased hepatic C18:0 concentration, while significantly decreased the concentrations of C16:1n-7, C20:1n-9, C18:3n-6, and C20:2n-9. Tl(III) exposure significantly reduced hepatic concentrations of C20:0, C22:0, C20:1n-9, C18:3n-6, and C20:3n-6. In addition, Tl(I) exposure upregulated the genes related to antioxidation (HO-1, GPX1, and GPX4), fatty acid synthesis (FADS2 and Elovl2), and fatty acid oxidation pathway (PPARα, ACADM, ACADVL, ACAA2, and CPT1A) in the liver. Tl(III) exposure did not significantly affect the transcript levels of liver antioxidative/metabolic enzymes and fatty acid synthesis-related genes, but upregulated fatty acid oxidation pathway-related genes (CYP4A10 and CPT1A). These results suggest that Tl(I) and Tl(III) exposures can cause liver damage and disrupt hepatic fatty acid metabolism, which provide new insights into Tl exposure-induced energy depletion from the perspective of fatty acid metabolism.

Effects of active immunization against a 13-amino acid receptor-binding epitope of FSHß on fertility regulation in female mice.

Follicle-stimulating hormone (FSH) is crucial for ovarian folliculogenesis and thus essential for female fertility. Here, we developed a novel FSH vaccine based on the tandem of a 13-amino acid receptor-binding epitope of FSHß (FSHß13AA-T) and used a mouse model to test its efficacy in female fertility regulation. Compared to placebo-immunized controls, FSHß13AA-T vaccination: induced a marked (P < 0.05) antibody generation; reduced (P < 0.05) serum concentrations of FSH, inhibin B and 17ß-estradiol; disrupted (P < 0.05) normal estrous cyclicity; delayed (P = 0.08) establishment of pregnancy; blocked (P < 0.05) folliculogenesis; and reduced (P < 0.05) litter size. Mechanistically, FSH vaccination reduced (P < 0.05) ovarian estrogen production by decreasing Lhcgr, Cyp19a1 and HSD3ß1 expression, and suppressed ovarian follicular development by decreasing ovarian Fshr, Inhα, Foxo3a, Bmp15 and Cdh1 expression. Overall, vaccination of female mice with FSHß13AA-T substantially disrupted FSH-dependent ovarian steroidogenesis and folliculogenesis, and caused subfertility. Therefore, vaccines based on FSHß13AA-T have potential as anti-fertility/contraceptive agents in females.