Combined collection systems of sewage and rainfall runoff seriously affect the spatial distributions of natural estrogens and their conjugates in river water: Insights from the Pearl River of China.

So far, little has been known about how the combined collection systems of sewage and rainfall runoff (CCSs) affect emerging contaminants in river water. To fill up the knowledge gap, this study was conducted to investigate the spatial distributions of three natural estrogens (NEs, i.e., estrone (E1), 17ß-estradiol (E2) and estriol (E3)) and their conjugates (C-NEs) in the Pearl River in the wet and dry seasons. Results showed that the respective average concentrations of NEs and C-NEs at different locations alongside the Pearl River in the wet season were 7.3 and 1.8 times those in the dry season. Based on estrogen equivalence (EEQ), the average estimated EEQ level in the Pearl River waters in the wet season was nearly 10 times that in the dry season. These seemed to imply that the CCSs in the wet season not only cause untreated sewage into the receiving water body, but greatly decrease the removal efficiency of NEs and C-NEs in wastewater treatment plant. Furthermore, the estimated annual loads of E1, E2, and E3 to the Pearl River in the wet season accounted for about 88.6 %, 100 %, and 99.3 % of the total annual loads. Consequently, this work for the first time demonstrated that the CCSs in cities with high precipitation are unfavorable for controlling of emerging contaminants.

Foxp2 deficiency impairs reproduction by modulating the hypothalamic-pituitary-gonadal axis in zebrafish†.

FOXP2 was initially characterized as a transcription factor linked to speech and language disorders. Single-cell RNA sequencing reveals that Foxp2 is enriched in the gonadotrope cluster of the pituitary gland and colocalized with the hormones LHB and FSHB in chickens and mice, implying that FOXP2 might be associated with reproduction in vertebrates. Herein, we investigated the roles of foxp2 in reproduction in a Foxp2-deficient zebrafish model. The results indicated that the loss of Foxp2 inhibits courtship behavior in adult male zebrafish. Notably, Foxp2 deficiency disrupts gonad development, leading to retardation of follicle development and a decrease in oocytes in females at the full-growth stage, among other phenotypes. The transcriptome analysis (RNA-seq) also revealed that differentially expressed genes clustered into the estrogen signaling and ovarian steroidogenesis-related signaling pathways. In addition, we found that Foxp2 deficiency could modulate the hypothalamic-pituitary-gonadal axis, especially the regulation of lhb and fshb expression, in zebrafish. In contrast, the injection of human chorionic gonadotropin, a specific LH agonist, partially rescues Foxp2-impaired reproduction in zebrafish, suggesting that Foxp2 plays an important role in the regulation of reproduction via the hypothalamic-pituitary-gonadal axis in zebrafish. Thus, our findings reveal a new role for Foxp2 in the regulation of reproduction in vertebrates.

Analysis and contamination levels of ten phthalic acid esters (PAEs) in Chinese commercial bubble tea: a comparison with commercial milk.

Phthalic acid ester (PAE) contamination in popular drink bubble tea has been hardly studied in the world. In this work, a liquid-liquid extraction following solid phase extraction (LLE-SPE)-UPLC-MS/MS method was first established for trace determination of ten PAEs in bubble tea. The developed method was validated with respect to linearity (R2 > 0.992), low limit of detections (LODs, 0.49-3.16 µg/L), and satisfactory recoveries (61.8-127.6%) with a low relative standard derivations (RSDs, 1.1-16.4%), which was also validated for commercial milk. Six out of ten PAEs, i.e., diethylhexyl phthalate (DEHP), dibutyl phthalate (DBP), diisobutyl phthalate (DIBP), diethyl phthalate (DEP), dihexyl phthalate (DHP), and diphenyl phthalate (DPP) were detected in Chinese bubble tea with concentrations ranging from not detection (ND) to 53.43 µg/L, while DEHP, DBP, DIBP, DEP, and dimethyl phthalate (DMP) were detected in commercial milk with concentrations ranging from ND to 110.58 µg/L. The respective average concentrations of DEHP in Chinese bubble tea and commercial milk were 19.40 and 23.46 µg/L, which were over two times that in drinking water quality standards of several countries including Israel, Korea, Oman, and Singapore (i.e., 8 µg/L). Calculated with human estimated daily intake (EDI), the average EDIs of five out of seven PAEs in bubble tea were higher than those in commercial milk. For example, the calculated EDI of DIBP in bubble tea was 5 times that in commercial milk, while their respective corresponding EDIs of DBP and DEHP were over 2.4 and 1.6 times. Based on estrogen equivalence (EEQ) with the unit of ng E2/L, the average EEQs of the ten PAEs in Chinese bubble tea and commercial milk were 14.26 and 17.06 ng E2/L, which were 52.8 and 62.3 times the observed effect concentration that could cause egg mortality of zebrafish. It is evident that the potential estrogenic effect of PAEs in bubble tea and commercial milk cannot be negligible. Given the fact that PAE contamination in bubble tea has been hardly investigated, such study is urgently to be performed in a global view.

Twelve natural estrogens and ten bisphenol analogues in eight drinking water treatment plants: Analytical method, their occurrence and risk evaluation.

Bisphenol analogues (BPs) and natural estrogens (NEs) as two important groups of endocrine-disrupting compounds (EDCs) in drinking water treatment plants (DWTPs) have been hardly investigated except bisphenol A (BPA) and three major NEs including estrone (E1), 17ß-estradiol (E2) and estriol (E3). In this study, a GC-MS analytical method was firstly established and validated for trace simultaneous determination of ten BPs and twelve NEs in drinking water, which included BPA, bisphenol B (BPB), bisphenol C (BPC), bisphenol E (BPE), bsiphenol F (BPF), bsiphenol P (BPP), bisphenol S (BPS), bisphenol Z (BPZ), bisphenol AF (BPAF), bisphenol AP (BPAP), E1, E2, E3, 17α-estradiol (17α-E2), 2-hydroestrone (2OHE1), 16hydroxyestrone (16α-OHE1), 4-hydroestrone (4OHE1), 2-hydroxyesstradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 17-epiestriol (17epiE3), 16-epiestriol (16epiE3) and 16keto-estraiol (16ketoE2). This investigation showed that eighteen out of twenty-two targeted compounds were detected in drinking source waters of eight DWTPs with concentrations ranging from not detected to 142.8 ng/L. Although the conventional treatment process of DWTP could efficiently remove both BPs and NEs with respective removal efficiencies of 74.1%-90.9% and 74.5%-100%, BPA, BPS, BPE, BPZ, E1, 2OHE1, and 2OHE2 were found in the finished drinking waters. Chlorination could remove part of BPs and NEs, but the efficiency varied greatly with DWTP and the reason was unknown. In the finished drinking waters of eight DWTPs, the highest chemically calculated estrogen equivalence (EEQ) derived from BPs and NEs was up to 6.11 ngE2/L, which was over 22 times that could do harm to zebrafish, indicating a potential risk to human health. Given the fact that many chlorination products of BPs and NEs likely have higher estrogenic activities, the estrogenic effect of BPs and NEs in finished drinking water should be accurately examined urgently with the inclusion of BPs, NEs as well as their main chlorinated by-products. This study shed new light on the occurrence, removal, and potential estrogenic effects of BPs and NEs in DWTPs.

Ten bisphenol analogues in Chinese fresh dairy milk: high contribution ratios of conjugated form, importance of enzyme hydrolysis and risk evaluation.

This study investigated concentration levels of ten bisphenols (BPs) in 13 Chinese commercial fresh low temperature dairy milk samples (fresh milk) of main local and national brands with or without enzyme hydrolysis. The results showed that at least two BPs were detected in each fresh milk sample without enzyme hydrolysis and the respective mean concentrations of bisphenol AF (BPAF), bisphenol B (BPB), bisphenol C (BPC), bisphenol F (BPF), bisphenol A (BPA), bisphenol S (BPS), bisphenol AP (BPAP), bisphenol PP (BPP), bisphenol Z (BPZ), and bisphenol E (BPE) were 0.73, 0.61, 1.86, 0.87, 0.42, 0.11, 1.06, 1.42, 1.5, and 0.04 ng/mL, while their respective detection frequencies ranged from 23.1-92.3%. These results indicated the frequent detection of BPs in fresh milk samples. With enzyme hydrolysis, the respective mean concentrations of BPAF, BPA, BPB, BPC, BPF, BPS, and BPAP were increased 7.1-107.1%, indicating the long-ignored importance of enzyme hydrolysis. The respective average estimated daily intakes (EDIs) of BPA by adult and children in China via fresh milk were 32.5 and 37.5 ng/kg bw/d, indicating that BPA in fresh milk was a crucial source to human. Six out of nine other BPs had higher average EDIs than that of BPA, among which the EDI of BPAP was almost three times that of BPA, suggesting the widespread contamination of other BPs in Chinese fresh milk.

Cholecystokinin (CCK) and its receptors (CCK1R and CCK2R) in chickens: functional analysis and tissue expression.

Cholecystokinin (CCK) is widely distributed in the gastrointestinal tract and central nervous system, regulating a range of physiological functions by activating its receptors (CCK1R and CCK2R). Compared to those in mammals, the CCK gene and its receptors have already been cloned in various birds, such as chickens. However, knowledge regarding their functionality and tissue expression is limited. In this study, we examined the expression of CCK and its 2 receptors in chicken tissues. In addition, the functionality of the 2 receptors was investigated. Using 3 cell-based luciferase reporter systems and western blots, we demonstrated that chicken (c-) CCK1R could be potently activated by cCCK-8S but not cCCK-4, whereas cCCK2R could be activated by cCCK-8S and cCCK-4 with similar efficiency. Using RNA-sequencing, we revealed that cCCK is abundantly expressed in the testis, ileum, and several brain regions (cerebrum, midbrain, cerebellum, hindbrain, and hypothalamus). The abundant expression of CCK in the hypothalamus was further supported by immunofluorescence. In addition, cCCK1R is highly expressed in the pancreas and moderately expressed in various intestinal regions (ileum, cecum, and rectum) and the pituitary gland, whereas cCCK2R expression is primarily restricted to the brain. Our data reveal the differential specificities of CCK receptors for various CCK peptides. In combination with the differential tissue distribution of CCK and its receptors, the present study helps to understanding the physiological functions of CCK/CCKRs in birds.

Simultaneous trace determination of three natural estrogens and their sulfate and glucuronide conjugates in municipal waste and river water samples with UPLC-MS/MS.

Analytical method for three natural estrogens (NEs) and their sulfate and glucuronide conjugates in waste and river waters using solid-phase extraction (SPE) coupled with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has been available, but problems including poor recovery exist. In order to solve these, some optimizations have been performed in this work. For sample preparation, both rinse and elution solutions were optimized, in which 6 mL of MeOH/water (1:9, v/v), MeOH/Ace/water (10:2:88, v/v/v), and MeOH/NH4OH/water (10:2:88, v/v/v) were determined as the rinse solution, while 6 mL of 2.0% NH4OH/MeOH was determined as the elution solution for conjugated NEs (C-NEs). For mobile phase, addition of NH4F could obviously enhance the signal response of the nine target compounds, and the optimized addition concentration was 0.5 mmol/L. The developed efficient method was validated and showed excellent linearity for each target compound (R2 > 0.998), low limit of quantifications (LOQs, 0.07-1.29 ng/L) in four different water matrices, and excellent recovery efficiencies of 81.0-116.1% in influent, effluent, ultra-pure, and river water samples with low relative standard deviations (RSDs, 0.6-13.6%). The optimized method was successfully applied to influent, effluent, and Pearl River water, among which three NEs were all detected, while five C-NEs were found in the influent, three C-NEs were detected in the effluent, and two C-NEs were found in the Pearl River water, indicating the wide distribution of NEs and C-NEs in different water environments. This work provided a reliable and efficient analytical method for simultaneous trace determination of NEs and C-NEs, which had satisfactory absolute recoveries with low RSDs, low LOQs, and time-saving for both analysis and nitrogen drying.

Characterization of CRH-Binding Protein (CRHBP) in Chickens: Molecular Cloning, Tissue Distribution and Investigation of Its Role as a Negative Feedback Regulator within the Hypothalamus-Pituitary-Adrenal Axis.

Corticotropin (ACTH) is a pituitary hormone playing important roles in stress response within the hypothalamus-pituitary-adrenal (HPA) axis. The biosynthesis and secretion of ACTH are controlled by multiple factors, including corticotropin-releasing hormone (CRH). As a key hypothalamus-derived regulator, CRH binds to corticotropin-releasing hormone receptor 1 (CRHR1) in the anterior pituitary gland to regulate ACTH synthesis and release. Thus, CRH-binding protein (CRHBP), which binds CRH with high affinity to inhibit CRH-induced ACTH secretion from pituitary cells, draws wide attention. In contrast to the extensive investigation of CRHBP in mammals and other lower vertebrates, the gene structure, tissue expression and physiological functions of CRHBP in birds remain largely unknown. In the present study, using chicken (c-) as our animal model, we examined the gene structure, tissue expression and functionality of CRHBP. Our results showed that: (1) cCRHBP cDNA encodes a 345 amino acid precursor, which shares high sequence identity with that of mammals, reptiles, frogs and fish; (2) cCRHBP is abundantly expressed in the brain (cerebrum and hypothalamus), pituitary and ovary; (3) cCRHBP inhibits the signaling of cCRHRs induced by cCRH, thus reducing the cCRH-induced ACTH secretion from cultured chick pituitary cells; (4) stress mediators (e.g., glucocorticoids) and stress significantly upregulate CRHBP mRNA expression in chickens, supporting its role as a negative feedback regulator in the HPA axis. The present study enriches our understanding of the conserved roles of CRHBP across vertebrates. In addition, chicken is an important poultry animal with multiple economic traits which are tightly controlled by the HPA axis. The characterization of the chicken CRHBP gene helps to reveal the molecular basis of the chicken HPA axis and is thus beneficial to the poultry industry.

Polystyrene Nanoplastic Exposure Induces Developmental Toxicity by Activating the Oxidative Stress Response and Base Excision Repair Pathway in Zebrafish (Danio rerio).

The widespread accumulation of nanoplastics is a growing concern for the environmental and human health. However, studies on the mechanisms of nanoplastic-induced developmental toxicity are still limited. Here, we systematically investigated the potential biological roles of nanoplastic exposure in zebrafish during the early developmental stage. The zebrafish embryos were subjected to exposure to 100 nm polystyrene nanoplastics with different concentrations (0, 100, 200, and 400 mg/L). The results indicated that nanoplastic exposure could decrease the hatching and survival rates of zebrafish embryos. In addition, the developmental toxicity test indicated that nanoplastic exposure exhibits developmental toxicity via the inhibition of the heart rate and body length in zebrafish embryos. Besides, behavioral activity was also significantly suppressed after 96 h of nanoplastic exposure in zebrafish larvae. Further biochemical assays revealed that nanoplastic-induced activation of the oxidative stress responses, including reactive oxygen species accumulation and enhanced superoxide dismutase and catalase activities, might affect developmental toxicity in zebrafish embryos. Furthermore, a quantitative polymerase chain reaction assay demonstrated that the mRNA levels of the base excision repair (BER) pathway-related genes, including lig1, lig3, polb, parp1, pold, fen1, nthl1, apex, xrcc1, and ogg1, were altered in zebrafish embryos for 24 h after nanoplastic exposure, indicating that the activation of the BER pathway would be stimulated after nanoplastic exposure in zebrafish embryos. Therefore, our findings illustrated that nanoplastics could induce developmental toxicity through activation of the oxidative stress response and BER pathways in zebrafish.

Evidence for Neuropeptide W Acting as a Physiological Corticotropin-releasing Inhibitory Factor in Male Chickens.

In vertebrates, adrenocorticotropin (ACTH), released by the pituitary gland, is a critical part of the stress axis and stress response. Generally, the biosynthesis and secretion of ACTH are controlled by both hypothalamic stimulatory factors and inhibitory factors [eg, ACTH-releasing inhibitory factor (CRIF)], but the identity of this CRIF remains unrevealed. We characterized the neuropeptide B (NPB)/neuropeptide W (NPW) system in chickens and found that NPW could directly target the pituitary to inhibit growth hormone (GH) and prolactin (PRL) secretion via neuropeptide B/W receptor 2 (NPBWR2), which is completely different from the mechanism in mammals. The present study first carried out a series of assays to investigate the possibility that NPW acts as a physiological CRIF in chickens. The results showed that (1) NPW could inhibit ACTH synthesis and secretion by inhibiting the 3',5'-cyclic adenosine 5'-monophosphate/protein kinase A signaling cascade in vitro and in vivo; (2) NPBWR2 was expressed abundantly in corticotrophs (ACTH-producing cells), which are located mainly in cephalic lobe of chicken pituitary, as demonstrated by single-cell RNA-sequencing, immunofluorescent staining, and fluorescence in situ hybridization; (3) dexamethasone could stimulate pituitary NPBWR2 and hypothalamic NPW expression in chicks, which was accompanied by the decease of POMC messenger RNA levels, as revealed by in vitro and subcutaneous injection assays; and (4) the temporal expression profiles of NPW-NPBWR2 pair in hypothalamus-pituitary axis and POMC in pituitary were almost unanimous in chicken. Collectively, these findings provide comprehensive evidence for the first time that NPW is a potent physiological CRIF in chickens that plays a core role in suppressing the activity of the stress axis.

Twelve natural estrogens in urines of swine and cattle: Concentration profiles and importance of eight less-studied.

Although four major natural estrogens (i.e., estrone (E1), 17ß-estradiol (E2), estriol (E3) and 17α-estradiol (αE2)) have been commonly found in livestock urine, this study reports the occurrence of eight other less-studied natural estrogens in urine of swine and cattle, i.e. 2-hydroxyestone (2OHE1), 4-hydroxyestrone (4OHE1), 2-hydroxyestradiol (2OHE2), 4-hydroxyestradiol (4OHE2), 16-epiestriol (16epiE3), 16α-hydroxyestrone (16αE1), 16-ketoestradiol (16ketoE2), and 17epiestriol (17epiE3). Results showed that each estrogen was found in at least one urine sample, and 6 of 8 the less-studied estrogens were present at frequencies of ≥90% in boars, ≥70% in sows, and ≥50% in dairy cattle. Five of eight the less-studied estrogens were present at frequencies of ≥33.3% in four beef cattle and one bull. On a concentration basis, the 8 less-studied natural estrogens represented 73.2%, 85.2%, 39.9%, 47.7%, 26.9%, 56.0% and 44.1% of total concentrations of the twelve natural estrogens when combining data from all animals. Similar results were observed based on estrogen equivalence, which indicated these newly detected eight less-studied natural estrogens were not negligible. This work is the first to figure out the importance of these less-studied natural estrogens in livestock urine, and their potential environmental risks associated with discharge of livestock wastewater should be urgently assessed in a holistic manner.

Stability properties of natural estrogen conjugates in different aqueous samples at room temperature and tips for sample storage.

It is important to keep natural estrogen conjugates (C-NEs) intact in aqueous environmental sample before sample preparation; otherwise, this may influence the accurate determination of NEs. Therefore, this work thoroughly investigated the stability of C-NEs in three different aqueous environmental samples under four different storage conditions, room temperature, low temperature of 4 °C, low pH of 3, and addition of HgCl2 at 2 g/L. Results showed that C-NEs in aqueous sample were easily deconjugated under low temperature of 4 °C, which has been widely used in sample collection and storage. Both the low pH of 3 and addition of HgCl2 at 2 g/L under room temperature could keep C-NEs intact in domestic wastewaters and river water within 36 h, but the latter could keep C-NEs stable longer. This work is the first to show that low pH of 3 alone could keep C-NEs intact, which suggested that the combined condition at low temperature of 4 °C that has been widely used could be omitted. Meanwhile, compared to pH adjustment, addition of 2 g/L HgCl2 into aqueous sample is more convenient and practical for 24-h composite sampling, which may be widely applied.

17α-ethynylestradiol and its two main conjugates in seven municipal wastewater treatment plants: Analytical method, their occurrence, removal and risk evaluation.

This work shows the existence of both 17-ethinylestradiol-3-sulfate (EE2-3S) and 17-ethinylestradiol-3-glucuronide (EE2-3G) in seven municipal WWTPs with substantial concentrations (n.d-50.10 ng/L). The calculated removal efficiencies of 17-ethinylestradiol (EE2) in the seven municipal WWTPs ranged from 40.8%-100% with an average removal efficiency of 83.3%. However, upon the inclusion of EE2 concentration transformed from EE2-3S and EE2-3G, the corresponding removal efficiencies were increased to 91.4%-100% with an average removal efficiency of 97.3%. This work is the first to clearly illustrate that EE2 conjugates in raw wastewater could greatly underestimate the removal effectiveness of municipal WWTPs on EE2, indicating the importance of the EE2 conjugates in municipal wastewater having been hardly paid with attention. The EE2-derived estrogen equivalence (EEQ) values in the effluents of seven WWTPs ranged from 0 to 0.98 ng E2/L having an average level of 0.45 ng E2/L, which were relatively low. However, upon the inclusion of EE2 transformable from EE2-3S and EE2-3G in effluents, the EE2-derived EEQ values in effluents would be increased to 0.77-4.85 ng E2/L having an average level of 2.71 ng E2/L, which clearly suggested that ignorance of EE2 conjugates in effluent would largely underestimate EE2's environmental risk to receiving water bodies.

Neuropeptide S (NPS) and its receptor (NPSR1) in chickens: cloning, tissue expression, and functional analysis.

Neuropeptide S (NPS) and its receptor neuropeptide S receptor 1 (NPSR1) have been suggested to regulate many physiological processes in the central nervous system (CNS), such as arousal, anxiety, and food intake in mammals and birds, however, the functionality and tissue expression of this NPS-NPSR1 system remain unknown in birds. Here, we cloned NPS and NPSR1 cDNAs from the chicken brain and reported their functionality and tissue expression. The cloned chicken NPS is predicted to encode a mature NPS peptide of 20 amino acids, which shows a remarkable sequence identity (∼94%) among tetrapod species examined, while NPSR1 encodes a receptor of 373 amino acids conserved across vertebrates. Using cell-based luciferase reporter systems, we demonstrated that chicken NPS could potently activate NPSR1 expressed in vitro and thus stimulates multiple signaling pathways, including calcium mobilization, cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways, indicating that NPS actions could be mediated by NPSR1 in birds. Quantitative real-time PCR revealed that NPS and NPSR1 are widely expressed in chicken tissues, including the hypothalamus, and NPSR1 expression is likely controlled by a promoter upstream exon 1, which shows strong promoter activities in cultured DF-1 cells. Taken together, our data provide the first proof that the avian NPS-NPSR1 system is functional and helps to explore the conserved role of NPS and NPSR1 signaling in tetrapods.

Single-Cell RNA Sequencing Analysis of Chicken Anterior Pituitary: A Bird's-Eye View on Vertebrate Pituitary.

It is well-established that anterior pituitary contains multiple endocrine cell populations, and each of them can secrete one/two hormone(s) to regulate vital physiological processes of vertebrates. However, the gene expression profiles of each pituitary cell population remains poorly characterized in most vertebrate groups. Here we analyzed the transcriptome of each cell population in adult chicken anterior pituitaries using single-cell RNA sequencing technology. The results showed that: (1) four out of five known endocrine cell clusters have been identified and designated as the lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs, respectively. Somatotrophs were not analyzed in the current study. Each cell cluster can express at least one known endocrine hormone, and novel marker genes (e.g., CD24 and HSPB1 in lactotrophs, NPBWR2 and NDRG1 in corticotrophs; DIO2 and SOUL in thyrotrophs, C5H11ORF96 and HPGDS in gonadotrophs) are identified. Interestingly, gonadotrophs were shown to abundantly express five peptide hormones: FSH, LH, GRP, CART and RLN3; (2) four non-endocrine/secretory cell types, including endothelial cells (expressing IGFBP7 and CFD) and folliculo-stellate cells (FS-cells, expressing S100A6 and S100A10), were identified in chicken anterior pituitaries. Among them, FS-cells can express many growth factors, peptides (e.g., WNT5A, HBEGF, Activins, VEGFC, NPY, and BMP4), and progenitor/stem cell-associated genes (e.g., Notch signaling components, CDH1), implying that the FS-cell cluster may act as a paracrine/autocrine signaling center and enrich pituitary progenitor/stem cells; (3) sexually dimorphic expression of many genes were identified in most cell clusters, including gonadotrophs and lactotrophs. Taken together, our data provides a bird's-eye view on the diverse aspects of anterior pituitaries, including cell composition, heterogeneity, cell-to-cell communication, and gene expression profiles, which facilitates our comprehensive understanding of vertebrate pituitary biology.

Veterinary antibiotics in swine and cattle wastewaters of China and the United States: Features and differences.

Veterinary antibiotics (VAs) have been widely used in livestock for disease prevention, treatment, and growth promotion. This study compared top 20 VAs in Chinese and US swine and cattle wastewater with published literatures. The sulfonamides (SAs) were found to be predominant, accounting for 62% of the top 20 VAs in Chinese swine wastewater, while tetracyclines (TCs) contributed to about 68.7% of the 18 VAs in US swine wastewater. The average concentration of the 20 major VAs in Chinese swine wastewater was estimated to be 1145 µg/L against 253.6 µg/L in the United States. On the other hand, the five major VAs in Chinese cattle wastewater were identified to be oxytetracycline, nafcillin, apramycin, lincomycin, and amikacin, while monensin was found to be dominant in US cattle wastewater. The average concentration of the top 20 VAs in Chinese and US cattle wastewaters were found to be 54.6 and 46.2 µg/L, respectively. These analyses suggested that VAs were probably over-used in Chinese swine industry, eventually causing the development and spreading of antibiotic resistant-bacteria and genes, which should be paid with attention. PRACTITIONER POINTS: Major veterinary antibiotics (VAs) in swine and cattle wastewater were identified. Top 20 VAs in swine and cattle wastewater of China and the United States were compared. VAs concentration in Chinese swine wastewater was 4.52 times that in the United States. VAs concentration in Chinese cattle wastewater was 1.18 times that of the United States.

Arginine vasotocin (AVT)/mesotocin (MT) receptors in chickens: Evidence for the possible involvement of AVT-AVPR1 signaling in the regulation of oviposition and pituitary prolactin expression.

Two structurally related peptides, arginine vasotocin (AVT) and mesotocin (MT), are reported to regulate many physiological processes, such as anti-diuresis and oviposition in birds, and their actions are likely mediated by four AVT/MT receptors (AVPR1A, AVPR1B, MTR and AVPR2b), which are orthologous/paralogous to human AVPR1A, AVPR1B, OXTR and AVPR2 respectively. However, our knowledge regarding the functions of these avian AVT/MT receptors has been limited. Here, we examined the functionality and expression of these receptors in chickens and investigated the roles of AVT in the anterior pituitary. Our results showed that 1) AVPR1A, AVPR1B and AVPR2b could be preferentially activated by AVT, monitored by cell-based luciferase reporter assays and/or Western blot, indicating that they are AVT-specific receptors (AVPR1A; AVPR1B) or AVT-preferring receptor (AVPR2b) functionally coupled to intracellular calcium, MAPK/ERK and cAMP/PKA signaling pathways. In contrast, MTR could be activated by AVT and MT with similar potencies, indicating that MTR is a receptor common for both peptides; 2) Using qPCR, differential expression of the four receptors was found in chicken tissues including the oviduct and anterior pituitary. In particular, only AVPR1A is abundantly expressed in the uterus, suggesting its involvement in mediating AVT-induced oviposition. 3) In cultured chick pituitary cells, AVT could stimulate ACTH and PRL expression and secretion, an action likely mediated by AVPR1B and/or AVPR1A abundantly expressed in anterior pituitary. Collectively, our data helps to elucidate the roles of AVT/MT in birds, such as the 'oxytocic action' of AVT, which induces uterine muscle contraction during oviposition.

The orphan G protein-coupled receptor 25 (GPR25) is activated by Apelin and Apela in non-mammalian vertebrates.

G protein-coupled receptor 25 (GPR25) is an orphan G protein-coupled receptor in vertebrates, that has been implicated to be associated with autoimmune diseases and regulate blood pressure in humans. However, the endogenous ligand of GPR25 remains unknown in vertebrates. Here, we reported that in non-mammalian vertebrates (zebrafish, spotted gars, and pigeons), GPR25 could be activated by Apelin and Apela peptides, which are also the two endogenous ligands of vertebrate Apelin receptor (APLNR). Using the pGL3-CRE-luciferase reporter assay and confocal microscopy, we first demonstrated that like APLNR, zebrafish GPR25 expressing in HEK293 cells could be effectively activated by zebrafish Apelin and Apela peptides, leading to the inhibition of forskolin-stimulated cAMP production and receptor internalization. Like zebrafish GPR25, pigeon and spotted gar GPR25 could also be activated by Apelin and Apela, and their activation could inhibit forskolin-induced cAMP accumulation. Interestingly, unlike zebrafish (/spotted gar/pigeon) GPR25, human GPR25 could not be activated by Apelin and Apela under the same experimental conditions. RNA-seq analysis further revealed that GPR25 is expressed in a variety of tissues, including the testes and intestine of zebrafish/spotted gars/humans, implying the potential roles of GPR25 signaling in many physiological processes in vertebrates. Taken together, our data not only provides the first proof that the orphan receptor GPR25 possesses two potential ligands 'Apelin and Apela' and its activation decreases intracellular cAMP levels in non-mammalian vertebrates, but also facilitates to unravel the physiological roles of GPR25 signaling in vertebrates.

Characterization of neuromedin U (NMU), neuromedin S (NMS) and their receptors (NMUR1, NMUR2) in chickens.

Neuromedin U (NMU) and its structurally-related peptide, neuromedin S (NMS), are reported to regulate many physiological processes and their actions are mediated by two NMU receptors (NMUR1, NMUR2) in mammals. However, the information regarding NMU, NMS, and their receptors is limited in birds. In this study, we examined the structure, functionality, and expression of NMS, NMU, NMUR1 and NMUR2 in chickens. The results showed that: 1) chicken (c-) NMU cDNA encodes a 181-amino acid precursor, which may generate two forms of NMU peptide with 9 (cNMU-9) and 25 amino acids (cNMU-25), respectively. 2) Interestingly, two cNMS transcripts encoding two cNMS precursors of different lengths were identified from chicken pituitary, and both cNMS precursors may produce a mature cNMS peptide of 9 amino acids (cNMS-9). 3) cNMU-9, cNMU-25 and cNMS-9 could activate cNMUR1 expressed in HEK293 cells potently, as monitored by three cell-based luciferase reporter systems, indicating that cNMUR1 can act as a receptor common for cNMU and cNMS peptides, whereas cNMUR2 could be potently activated by cNMS-9, but not by cNMU-9/cNMU-25. 4) cNMU and cNMUR1 are widely expressed in chicken tissues with abundant expression noted in the gastrointestinal tract, as detected by quantitative real-time PCR, whereas cNMUR2 expression is mainly restricted to the brain and anterior pituitary, and cNMS is widely expressed in chicken tissues. Collectively, our data helps to elucidate the physiological roles of NMU/NMS peptides in birds and reveal the functional conservation and changes of NMU/NMS-NMUR axis across vertebrates.

Characterization of the Apelin/Elabela Receptors (APLNR) in Chickens, Turtles, and Zebrafish: Identification of a Novel Apelin-Specific Receptor in Teleosts.

Apelin receptor(s) (APLNR) are suggested to mediate the actions of apelin and Elabela (ELA) peptides in many physiological processes, including cardiovascular development and food intake in vertebrates. However, the functionality of APLNR has not been examined in most vertebrate groups. Here, we characterized two APLNRs APLNR1, APLNR2) in chickens and red-eared sliders, and three APLNRs in zebrafish (APLNR2a, APLNR2b, APLNR3a), which are homologous to human APLNR. Using luciferase-reporter assays or Western blot, we demonstrated that in chickens, APLNR1 (not APLNR2) expressed in HEK293 cells was potently activated by chicken apelin-36 and ELA-32 and coupled to Gi-cAMP and MAPK/ERK signaling pathways, indicating a crucial role of APLNR1 in mediating apelin/ELA actions; in red-eared sliders, APLNR2 (not APLNR1) was potently activated by apelin-36/ELA-32, suggesting that APLNR2 may mediate apelin/ELA actions; in zebrafish, both APLNR2a and APLNR2b were potently activated by apelin-36/ELA-32 and coupled to Gi-cAMP signaling pathway, as previously proposed, whereas the novel APLNR3a was specifically and potently activated by apelin. Similarly, an apelin-specific receptor (APLNR3b) sharing 57% sequence identity with zebrafish APLNR3a was identified in Nile tilapia. Collectively, our data facilitates the uncovering of the roles of APLNR signaling in different vertebrate groups and suggests a key functional switch between APLNR1 and APLNR2/3 in mediating the actions of ELA and apelin during vertebrate evolution.