Estrogen Receptor Gene 1 (ESR1) Mediates Lipid Metabolism in Goose Hierarchical Granulosa Cells Rather than in Pre-Hierarchical Granulosa Cells.

(1) Background: The role of estrogen receptor gene 1 (ESR1) in female reproduction and lipid metabolism has been extensively investigated. However, its contribution to lipid metabolism during the development of poultry follicles remains unclear. (2) Methods: This study aimed to explore the function of ESR1 via overexpressing (ESR1ov) and interfering (ESR1si) with its expression in pre-hierarchical granulosa cells (phGCs) and hierarchical granulosa cells (poGCs). (3) Results: We successfully cloned and obtained an 1866 bp segment of the full-length CDS region of the Sichuan white goose ESR1 gene. In phGCs of the ESR1ov and ESR1si groups, there were no significant changes compared to the control group. However, in poGCs, the ESR1ov group exhibited decreased lipid deposition, triglycerides, and cholesterol compared to the control group, while the ESR1si group showed increased lipid deposition, triglycerides, and cholesterol. The expression of APOB and PPARα was significantly reduced in the ESR1ov group compared to the ESR1ov-NC group. Moreover, significant changes in the expression of ACCα, DGAT1, SCD, CPT1, and ATGL were observed between the ESR1si and ESR1si-NC group. (4) Conclusions: These findings shed light on the function and molecular mechanism of ESR1 in lipid metabolism in goose poGCs, providing a better understanding of the physiological process of goose follicular development.

MiR-202-5p Regulates Geese Follicular Selection by Targeting BTBD10 to Regulate Granulosa Cell Proliferation and Apoptosis.

The regulation of granulosa cells (GCs) proliferation and apoptosis is the key step in follicular selection which determines the egg production performance of poultry. miR-202-5p has been reported to be involved in regulating the proliferation and apoptosis of mammalian ovarian GCs. However, its role in regulating the proliferation and apoptosis of goose GCs is still unknown. In the present study, the GCs of pre-hierarchical follicles (phGCs, 8-10 mm) and those of hierarchical follicles (hGCs, F2-F4) were used to investigate the role of miR-202-5p in cell proliferation and apoptosis during follicle selection. In phGCs and hGCs cultured in vitro, miR-202-5p was found to negatively regulate cell proliferation and positively regulate cell apoptosis. The results of RNA-seq showed that BTB Domain Containing 10 (BTBD10) is predicted to be a key target gene for miR-202-5p to regulate the proliferation and apoptosis of GCs. Furthermore, it is confirmed that miR-202-5p can inhibit BTBD10 expression by targeting its 3'UTR region, and BTBD10 was revealed to promote the proliferation and inhibit the apoptosis of phGCs and hGCs. Additionally, co-transfection with BTBD10 effectively prevented miR-202-5p mimic-induced cell apoptosis and the inhibition of cell proliferation. Meanwhile, miR-202-5p also remarkably inhibited the expression of Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta (PIK3CB) and AKT Serine/Threonine Kinase 1 (AKT1), while it was significantly restored by BTBD10. Overall, miR-202-5p suppresses the proliferation and promotes the apoptosis of GCs through the downregulation of PIK3CB/AKT1 signaling by targeting BTBD10 during follicular selection. Our study provides a theoretical reference for understanding the molecular mechanism of goose follicular selection, as well as a candidate gene for molecular marker-assisted breeding to improve the geese' egg production performance.

miR-202-5p Inhibits Lipid Metabolism and Steroidogenesis of Goose Hierarchical Granulosa Cells by Targeting ACSL3.

miRNAs are critical for steroidogenesis in granulosa cells (GCs) during ovarian follicular development. We have previously shown that miR-202-5p displays a stage-dependent expression pattern in GCs from goose follicles of different sizes, suggesting that this miRNA could be involved in the regulation of the functions of goose GCs; therefore, in this study, the effects of miR-202-5p on lipid metabolism and steroidogenesis in goose hierarchical follicular GCs (hGCs), as well as its mechanisms of action, were evaluated. Oil Red O staining and analyses of intracellular cholesterol and triglyceride contents showed that the overexpression of miR-202-5p significantly inhibited lipid deposition in hGCs; additionally, miR-202-5p significantly inhibited progesterone secretion in hGCs. A bioinformatics analysis and luciferase reporter assay indicated that Acyl-CoA synthetase long-chain family member 3 (ACSL3), which activates long-chain fatty acids for the synthesis of cellular lipids, is a potential target of miR-202-5p. ACSL3 silencing inhibited lipid deposition and estrogen secretion in hGCs. These data suggest that miR-202-5p exerts inhibitory effects on lipid deposition and steroidogenesis in goose hGCs by targeting the ACSL3 gene.

miR-375 upregulates lipid metabolism and inhibits cell proliferation involved in chicken fatty liver formation and inheritance via targeting recombination signal binding protein for immunoglobulin kappa J region (RBPJ).

Poultry is susceptible to fatty liver which lead to decrease egg production and increase mortality. But the potential molecular mechanisms remain largely unclear. In the current study, in combination with transcriptome sequencing and miRNA sequencing data analysis from F1 generation of the normal liver and fatty liver tissues, the differentially expressed miR-375 and its target gene RBPJ were screened and verified. The expression levels of miR-375 and RBPJ gene in the liver between control and fatty liver groups of F0-F3 generation for Jingxing-Huang (JXH) chicken are different significantly (P < 0.05 or P < 0.01). And downregulated RBPJ expression can promote TG content and lipid droplets in primary hepatocytes cultured in vitro (P < 0.01). Cell proliferation-related genes, including PMP22, IGF-1, IGF-2, and IGFBP-5, increased or decreased significantly after overexpression or knock-down RBPJ (P < 0.05 or P < 0.01), respectively. This study uniquely revealed that miR-375 induced lipid synthesis and inhibited cell proliferation may partly due to regulation of RBPJ expression, thereby involving in fatty liver formation and inheritance in chicken. The results could be useful in identifying candidate genes and revealing the pathogenesis of fatty liver that may be used for disease-resistance selective breeding in chicken.

De novo assembly of a wild swan goose (Anser cygnoides) genome.

The swan goose (Anser cygnoides) is the ancestor of the Chinese domestic goose. A previous study reported a scaffold-level genome version for a Chinese indigenous goose breed, and this assembly was used as the swan goose's reference genome. To date, there is still a lack of a chromosome-level genome for the swan goose. Here, we reported a de novo assembly of the genome of a wild swan goose using an integrated strategy that combines Illumina Hiseq, Oxford Nanopore and chromosome conformation capture (Hi-C) sequencing. A total of 134.6 Gb Nanopore data with sequencing coverage of 110.33 and 69.45 Gb Illumina data with coverage of 56.93 were obtained. The genome assembly size was 1153.41 Mb, with a contig N50 of 22.75 Mb. The total size and N50 length of our assembly were larger than the previously reported scaffold-level genome version. In addition, whole-genome sequencing data of 10 geese were mapped to the previous and the current assemblies. On average, 97.88 and 93.18% of the reads were properly mapped and paired into our and the previous assemblies. This high-quality chromosome-level swan goose genome could provide a valuable resource for the utilisation of goose studies and breeding.

Comparative Analyses of Production Performance, Meat Quality, and Gut Microbial Composition between Two Chinese Goose Breeds.

Goose meat is consumed by consumers because it contains a relatively high proportion of polyunsaturated fatty acids (PUFAs). This study was conducted to explore the main differences in production performance, breast meat quality traits, and cecal microbiota compositions between the Zi goose (ZG) and Xianghai flying goose (FG). The production performance and breast meat quality trait analyses showed that compared with the ZG, the FG had a higher right breast muscle index, ileum villi height/crypt depth ratio (VH/CD), and cecum fermentation rate (higher short-chain fatty acid (SFCA) concentration); a lower abdominal fat index; a higher proportion of PUFAs; and a lower shear force. Spearman's correlation coefficients between the cecal microbiota composition and production performance indexes suggested that the genus Faecalibacterium was positively associated with production performance; in contrast, the genus Candidatus Saccharimonas was negatively correlated with production performance; moreover, the Ruminococcus torques group, Parasutterella, and Methanobrevibacter were negatively related to the VH/CD. Taken together, in this particular trial, FG had better production performance, healthier meat quality traits, and better intestinal digestion and absorption capacities than ZG. These results not only provide a useful data reference for the production of healthy geese for human consumption but can also help guide the utilization of goose breed resources.

miR-375 Induced the Formation and Transgenerational Inheritance of Fatty Liver in Poultry by Targeting MAP3K1.

The liver of poultry is the primary site of lipid synthesis. The excessive production of lipids accumulates in liver tissues causing lipid metabolism disorders, which result in fatty liver disease and have a transgenerational effect of acquired phenotypes. However, its specific mechanisms have not yet been fully understood. In this study, the differentially expressed miR-375 as well as its target gene MAP3K1 (mitogen-activated protein kinase kinase kinase 1) were screened out by interaction network analysis of microRNA sequencing results and transcriptome profiling in the fatty liver group of the F0-F3 generation (p < 0.05 or p < 0.01). Furthermore, the results showed that the number of lipid droplets and triglyceride content were significantly decreased after upregulation of miR-375 in primary hepatocyte culture in vitro (p < 0.05 or p < 0.01). The MAP3K1 knockdown group exhibited the opposite trends (p < 0.05 or p < 0.01). P53, Bcl-x, PMP22, and CDKN2C related to cell proliferation were significantly upregulated or downregulated after knocking down MAP3K1 (p < 0.05). This research uniquely revealed that silencing miR-375 inhibits lipid biosynthesis and promotes cell proliferation, which may be due to the partial regulation of the expression level of MAP3K1, thereby further participating in the transgenerational inheritance process of regulating liver lipid metabolism. These results reveal the pathogenesis of fatty liver in noncoding RNA and provide good candidate genes for breeding progress of disease resistance in chickens.

CircRNA-WNK2 Acts as a ceRNA for miR-328a-3p to Promote AANAT Expression in the Male Rat Pineal Gland.

Noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and circular RNAs (circRNAs), which are expressed with a daily rhythm in the rat pineal gland, are associated with the regulation of melatonin secretion and other biological functions. However, the mechanisms of these molecules in the rat pineal gland are not yet fully understood. In this study, we found that circR-WNK2 was highly expressed at night, which may be involved in the regulation of melatonin secretion through the competitive endogenous RNA (ceRNA) mechanism. By dual luciferase reporter, RNA pull-down, and fluorescence in situ hybridization (FISH) assays, we found that miR-328a-3p can target circR-WNK2 and the Aa-nat mRNA 3'UTR. Transfection experiments indicated that circR-WNK2 could competitively bind to miR-328a-3p, reduce miR-328a-3p expression, and promote Aa-nat gene expression and melatonin secretion. And by constructing a superior cervical ganglionectomy (SCGx) rat model, we found that ncRNAs expression in the pineal gland was regulated by signals from the suprachiasmatic nucleus. This finding supports the hypothesis that these noncoding RNAs may interact to shape the circadian rhythm through transcriptional processing in melatonin synthesis.

GMP production of [18F]FDOPA and issues concerning its quality analyses as in USP "Fluorodopa F 18 Injection".

INTRODUCTION: Lately, 6-[(18)F]fluoro-L: -DOPA (FDOPA) has found increase in its clinical demand for whole-body positron emission tomography (PET) scans, and two key issues in fulfilling this demand are the difficulties in producing FDOPA under the recently imposed PET drug good manufacturing practice (GMP) regulations and in providing it in the quality meeting the terms of major compendia. This paper describes the approaches for the GMP production of FDOPA and for the product testing to meet the standard of United States Pharmacopeia (USP) "Fluorodopa F 18 Injection." METHODS: FDOPA was produced by the carrier-added electrophilic aromatic substitution reaction in the facility complying Pharmaceutical Inspection Cooperation Scheme clean room standard. The special aseptic handling technique was applied to minimize the bioburden. The product quality control followed all testing items and procedures, including three different settings of HPLC. RESULTS: The process yielded FDOPA average 2.60 ± 0.26 GBq (N = 22) in every batch. All qualities of the product were within the specifications described in the USP "Fluorodopa F 18 Injection." The entire production was audited by the government authority and certified to comply with the latest PET drug GMP regulation. CONCLUSION: Our efforts in producing FDOPA following all aspects of GMP requirements have resulted in a product with the USP quality and certified as GMP complied. The routine production yields enough doses for three to four whole-body scans in each batch. The issues discussed in the report provide good reference for producers planning in routine production for PET drugs that are not commonly produced or with complicated compendial quality control tests.

An efficient and aseptic preparation of "sodium fluoride ((18)F) injection" in a GMP compliant facility.

INTRODUCTION: "Sodium fluoride ((18)F) injection" is an isotonic NaCl solution containing [(18)F]NaF to be used as bone imaging agent. Although its NDA was approved by the US FDA in 1972, it has not been commercially available since 1975 due to mostly the popularity of (99m)Tc-MDP. Recently, advances in PET/CT technology and the often interrupted (99m)Tc supply have led to the renewed interest in the use of [(18)F]NaF to detect bone metastases in cancer patients. This report introduces an efficient, low-cost and aseptic preparation of "Sodium fluoride ((18)F) injection" for PET scan. METHOD: (18)F-Fluoride in target water from cyclotron was adsorbed onto four different forms of anion-exchange resins then desorbed by isotonic NaCl solution into the product vial. One of the resins that yielded the product at the suitable pH was used for the aseptic preparation. The components for this setup, including stopcocks, extension tubes, etc., were all single-use, individually packed and sterile. The process was done in a lead-line isolator maintained in grade A (PIC/S) aseptic condition. The quality of the obtained "Sodium fluoride ((18)F) injection" was analyzed according to its monograph in the European Pharmacopoeia (EP). RESULTS: The resin in the chloride form yielded the product of pH 6.7 and was chosen for the subsequent preparation. The radiochemical yield was quantitative. The product met all criteria specified in EP, including biological, physical and chemical specifications. CONCLUSIONS: This method is an efficient, space-saving and extremely low-cost operation that easily performed in an aseptic environment meeting GMP standard. The quality of the "Sodium fluoride ((18)F) injection" so yielded meets EP specifications. This setup provides hospital with facility meeting GMP standard a cost effective and efficient method for "Sodium fluoride ((18)F) injection" production without the need for the expensive automatic module and extra QC instrument.

[18F]FBAU 3',5'-dibenzoate, a lipophilic prodrug, enhances brain uptake of the cell proliferation tracer [18F]FBAU.

INTRODUCTION: 1-(2-deoxy-2-[(18)F]fluoro-beta-D-arabinofuranosyl)-5-bromouracil ([(18)F]FBAU) is a cell proliferation tracer. However, it does not pass readily through the blood-brain barrier. We synthesized a lipophilic prodrug of [(18)F]FBAU that was intended to enhance brain uptake of [(18)F]FBAU to improve the imaging of brain cell proliferation. METHODS: [(18)F]FBAU was synthesized according to the methods described by Alauddin [J Med Chem 39 (1996) 2835-2843]. The prodrug, 1-(2-deoxy-3,5-O-dibenzoyl-2-[(18)F]fluoro-beta-D-arabinofuranosyl)-5-bromouracil ([(18)F]FBAU 3',5'-dibenzoate), was purified from an intermediate of [(18)F]FBAU. Their lipophilicity was determined by performing octanol/water partition coefficient (log P) measurements. In vitro metabolic fates of the prodrug were examined in rat and mouse plasma and brain homogenates. Brain uptake was determined following iv injection of the radiotracers by killing animals at various time points and dissecting and counting the radioactivity accumulation in the various tissues. RESULTS: Values of log P for [(18)F]FBAU 3',5'-dibenzoate and [(18)F]FBAU were 3.95 and -0.35, respectively. In rat plasma, the prodrug was gradually hydrolyzed to [(18)F]FBAU. Thirty minutes after mixing [(18)F]FBAU 3',5'-dibenzoate in the plasma, 25% of the prodrug had been hydrolyzed. The hydrolysis went more slowly in brain homogenates. At 15 min post injection, relative to animals injected with [(18)F]FBAU, brain uptake of radioactivity in animals injected with [(18)F]FBAU 3',5'-dibenzoate was increased by 150% (P=.005) and 78% (P=.037) in rats and mice, respectively. At 60 min post injection, the radioactive contents extracted from the brain were mostly [(18)F]FBAU. CONCLUSION: The synthesized novel prodrug [(18)F]FBAU 3',5'-dibenzoate has enhanced brain uptake in rodents, suggesting it may be useful as an imaging agent for tracing brain cell proliferation.