Golden pompano (Trachinotus ovatus) is an economically important marine aquaculture species, but its production is frequently threatened by bacterial diseases, particularly infection by Streptococcus iniae. Caspases are central regulators of apoptosis and inflammatory signaling, yet their functional diversification and regulatory mechanisms in T. ovatus remain poorly understood. In this study, we systematically characterized the caspase gene family in T. ovatus and selected Tovcasp3a and Tovcasp8a for further investigation based on their infection-responsive and phenotype-associated expression patterns. Expression analyses showed that Tovcasp3a was associated with modulation of intrinsic apoptotic signaling, whereas Tovcasp8a was linked to activation of RIPK1/FADD/NF-κB-related inflammatory responses. In situ hybridization further localized both transcripts predominantly to hepatic vascular wall cells. Promoter dissection and CpG methylation analyses indicated that upstream cis-regulatory elements and DNA methylation contribute to the differential expression of these two genes. Collectively, these results support a coordinated regulatory model in which apoptosis and inflammatory signaling are balanced during host defense against S. iniae. This study provides new insight into the molecular basis of antibacterial immunity in golden pompano and identifies candidate regulatory targets for future disease-resistance research.

Emerging evidence suggests that goat milk exerts beneficial effects on insulin levels, insulin sensitivity, and glucose homeostasis in type 2 diabetes mellitus (T2DM). However, the underlying mechanisms need further investigation. Here, the metabolic profiles of goat milk, goat milk powder, and cow milk were compared, revealing distinct compositional signatures. In a high-fat diet/streptozotocin-induced T2DM mouse model, goat milk intervention significantly improved glucose homeostasis (fasting blood glucose decreased from 13.87 mmol/L to 8.08 mmol/L, P < 0.05), alleviated hepatic injury, and remodeled serum bile acid metabolism. Further investigation revealed that goat milk intervention altered the gut microbiota composition, characterized by an increased relative abundance of Akkermansia and Lachnoclostridium. Additionally, transcriptomic analysis suggested that goat milk intervention up-regulated Bcl-2 and PI3K and down-regulated G6pc in the hepatic PI3K/AKT pathway. Collectively, these findings suggest that goat milk has the potential to alleviate T2DM and may serve as a dietary intervention for T2DM patients.

OBJECTIVE:Fatty acids and calcium are both important nutrients in goat milk. Investigating the upstream molecular regulatory mechanisms that control the synthesis of milk fat and milk calcium in the mammary gland can help improve the quality of milk at its source. The objective of this study was to investigate the effects and regulatory pathways of serotonin (5-hydroxytryptamine, 5-HT) and its receptors on lipid synthesis and calcium ion levels in goat mammary epithelial cells (GMECs). METHODS:GMECs isolated from live goats were treated with serotonin, overexpression of Serotonin receptor 2A (HTR2A), Sarpogrelate ([SAR] the specific antagonist of HTR2A), or a combination of these agents. The expression of genes related to de novo lipid synthesis in GMECs were detected using the Quantitative Real-Time polymerase chain reaction, the content of lipid droplets was detected using the BODIPY assay, and the calcium content was detected using the calcium chelating probe Fluo-3AM assay. RESULTS:5-HT dose-dependently promotes the activity of GEMCs, significantly inhibits the mRNA expression of key genes involved in de novo lipid synthesis such as ACC, FASN, SREBP1, SCD1 and ELOVL6 at a concentration of 100 μM, reduces triglyceride and total cholesterol content, suppresses lipid droplet accumulation in cells, and simultaneously promotes calcium accumulation in cells. Furthermore, overexpression of HTR2A in GMECs also induces an increase in cellular calcium levels and inhibits lipid synthesis and accumulation in cells. However, treatment of cells with SAR, the specific antagonist of HTR2A, significantly increases the levels of triglycerides, total cholesterol, and lipid droplet accumulation in cells. CONCLUSION:5-HT inhibits lipid synthesis in GMECs while promoting an increase in cellular calcium levels, and this effect is mediated by the HTR2A receptor. Furthermorey, antagonists targeting HTR2A can reverse the inhibition of lipid synthesis and accumulation in cells.

Embryos contain a large number of lipid droplets, and lipid metabolism is gradually activated during embryonic development to provide energy. However, the regulatory mechanisms remain to be investigated. Stearoyl-CoA desaturase 1 (Scd1) is a fatty acid desaturase gene that is mainly involved in intracellular monounsaturated fatty acid production, which takes part in many physiological processes. Analysis of transcripts at key stages of embryo development revealed that Scd1 was important and expressed at an increased level during the cleavage and blastocyst stages. Knockout Scd1 gene by CRISPR/Cas9 from zygotes revealed a decrease in lipid droplets (LDs) and damage in the inner cell mass (ICM) formation of blastocyst. Comparative analysis of normal and knockout embryo transcripts showed a suppression of ribosome protein (RPs) genes, leading to the arrest of ribosome biogenesis at the 2-cell stage. Notably, the P53-related pathway was further activated at the blastocyst stage, which eventually caused embryonic development arrest and apoptosis. In summary, Scd1 helps in providing energy for embryonic development by regulating intra-embryonic lipid droplet formation. Moreover, deficiency activates the RPs-Mdm2-P53 pathway due to ribosomal stress and ultimately leads to embryonic development arrest. The present results suggested that Scd1 gene is essential to maintain healthy development of embryos by regulating energy support.

5-Hydroxytryptamine (5-HT) is an amine produced in both the mammary gland and the central nervous system. Tryptophan hydroxylase 1 (TPH1) catalyzes the conversion of 5-hydroxytryptophan (5-HTP) into l-tryptophan, which is then converted into 5-HT by monoamine-oxidase (MAO-A). In the mammary gland, 5-HT has been shown to have a variety of paracrine-autocrine actions, including suppressing lactation, controlling the destiny of mammary epithelial cells, and maintaining calcium homeostasis throughout the transition from pregnancy to lactation. To examine the effects of 5-HT on the composition of colostrum and milk, a total of 30 transition Guan Zhong dairy goats were intramuscularly injected with 5-HTP (1.0 mg/kg) every morning before feeding from 10 d before the projected parturition date to the day of parturition. The average number of days animals received injections was 8.2 ± 3.2 d. 5-HTP treatment increased serum 5-HT concentration from days 5 to 2 relative to parturition (P < 0.05), and decreased the casein concentration of colostrum (P < 0.05). In the in vitro experiment, mammary epithelial cells isolated from three individual goats' mammary glands were separately treated with 200 μM 5-HTP, 30 μM PCPA (the specific inhibitor of TPH1), or 200 μM 5-HTP + 50 μM SB269970 (the selective antagonist of 5-HTR7). The results showed that 200 μM 5-HTP inhibited the expression of β-casein, downregulated the activity of the JAK2/ STAT5a signaling pathway, and promoted the apoptosis of goat mammary epithelial cells (GMECs) (P < 0.05). When GMECs were treated with 30 μM Four-chloro-dl-phenylalanine (PCPA), a specific inhibitor of 5-HT synthesis, the mRNA expression of STAT5a and the phosphorylated STAT5a protein level were upregulated. The 50 μM SB269970 treatment rescued the effects of 5-HTP on GMECs (P < 0.05). Taken together, the results indicated that 5-HTP exerted an inhibitory effect on β-casein synthesis and a proapoptotic effect in GMECs via HTR7 and the JAK2/STAT5a axis.

SCOPE:Consuming goat milk is known to benefit high-fat diet-fed and streptozocin (STZ)-induced diabetic rats, but the underlying mechanisms are unknown. This study is conducted to investigate the metabolic effects of a goat milk diet (a form of goat milk powder) on glucose homeostasis and pancreatic conditions in a mouse model of Type 2 diabetes mellitus (T2DM) induced by STZ. METHODS AND RESULTS:T2DM mice are fed with a goat-milk-based diet containing 10.3% w/w goat milk powder for 10 weeks for investigating the in vivo effects; a β-cell line MIN6 cells are used to test the in vitro effects of digested goat milk (DGM). Goat milk diet improves the deleterious effects of STZ on fasting glucose levels and glucose tolerance, accelerates pancreatic structure recovery, and alters blood metabolites in mice. Based on the significant differences observed in metabolites, the key pathways, metabolite regulatory enzymes, metabolite molecular modules, and biochemical reactions are identified as critical integrated pathways. DGM promotes the cell activity, glucose transportation, and AKT activation in cultured STZ-treated MIN6 cells in vitro. CONCLUSIONS:Goat milk diet improves glucose homeostasis and pancreatic conditions of T2DM mice, in association with improved blood metabolite profiles and activation of pancreatic AKT pathway.

Due to the large amounts of calcium transferred to milk from mammary glands, periparturient dairy goats face challenges with calcium metabolism disorder and hypocalcemia. Serotonin (5-hydroxytryptamine, 5-HT), the product of 5-hydroxy-l-tryptophan (5-HTP) catalyzed by tryptophan hydroxylase 1, is a multifunctional monoamine thought to be a homeostatic regulator of the animal. The objective of the current study was to investigate the effects and underlying mechanisms of intramuscular 5-HTP injections on calcium homeostasis in the goat mammary glands. In the in vivo experiment, 30 multiparous Guanzhong dairy goats were randomly assigned to 2 groups, one group was injected with 5-HTP intramuscularly and the other group was injected with normal saline. From the first 10 d of the expected date for delivery, 5-HTP or saline was injected into goats through the shoulder muscle every morning before feeding, with a dose of 1 mg/kg per body weight. In the in vitro experiment, goat mammary epithelial cells (GMEC) were treated with 100 μM 5-HT for the evaluation of 5-HT in calcium transportation. The results demonstrated that 5-HTP treatment had no effect on the basic composition of colostrum (P > 0.05) but increased the serum 5-HT concentrations on days -5, -4, -3, and 5 relative to parturition (P < 0.05). The 5-HTP injection group had greater serum calcium concentration on day 4 and greater serum parathyroid hormone-related protein (PTHrP) on days -5, -4, -1, 3, 4, and 5 compared with the saline injection group (P < 0.05). It was further confirmed that 5-HT could increase intracellular calcium levels by increasing PTHrP and decreasing plasma membrane Ca2+-ATPases1 (PMCA1) in GMEC (P < 0.05). In conclusion, 5-HTP treatment in multiparous goats during the transition period from pregnancy to lactation is a feasible way to protect goats from calcium metabolism disorder.

Delta-5 desaturase (D5D), encoded by the fatty acid desaturase 1 (FADS1) gene, is a rate-limiting enzyme in polyunsaturated fatty acid (PUFA) synthesis that influences the PUFA levels in milk fat. However, the function and molecular mechanism of FADS1 in milk fat metabolism remain largely unknown. The FADS1 overexpression increased the triglyceride content, lipid droplet size, and expression of genes related to fatty acid de novo synthesis (SREBP1 and ACC), intracellular fatty acid transporters (FABP3 and FABP4) and triacylglycerol synthesis gene (DGAT2). It also significantly promoted the SREBP1 nuclear translocation by inhibiting the AMPK activation. In addition, FADS1 overexpression inhibited cell proliferation and arrested cell cycle at the G1 phase. These findings reveal a novel FADS1-AMPK-SREBP1 pathway regulating milk fat production in the goat mammary gland.

Background: Calcium is one of the major mineral nutrients in goat milk. Tryptophan hydroxylase1 (TPH1) is a rate-limiting enzyme catalyzing hydroxylation of L-tryptophan into 5-hydroxytryptamine (5-HT) essential for maintaining calcium homeostasis. The function of TPH1 and 5-HT in goat mammary calcium homeostasis is not well known.Methods: The CRISPR/Cas9-meidated TPH1 knockout goat mammary epithelial cells (GMEC) were constructed firstly. Then the content of 5-HT, intracellular calcium level and abundance of key genes related to calcium transportation were evaluated and compared in wild-type GMEC, TPH1 knockout GMEC, to explore the impact of TPH1 on calcium transportation, respectively. Wild-type GMEC and TPH1 knockout GMEC were further treated with exogenous 5-HTP to confirm the role of TPH1 in regulating calcium homeostasis in GMEC. 5-HT concentration was measured by enzyme-linked immunosorbent assay and fluo-3 staining was used to determine intracellular calcium content.Results: The TPH1 knockout GMEC heterozygous clone with no off-target effects was obtained after transfection of the Cas9/sgRNA expression vector. The 5-HT synthesis and intracellular calcium level decreased in TPH1 gene knockout GMEC. The mRNA abundance of secretory-pathway Ca2+ -ATPase1 (SPCA1) and plasma membrane Ca2+-ATPase1 (PMCA1) were up-regulated while the mRNA abundance of secretory-pathway Ca2+ -ATPase2 (SPCA2) was down-regulated in TPH1 knockout GMEC. Up-regulation of parathyroid hormone-related peptide (PTHrP), a key regulator of mammary calcium metabolism, induced by 5-HTP were blocked by TPH1 gene knockout. The TPH1 knockout GMEC showed a lower sensitivity to 5-HTP induced elevation of calcium content.Conclusion: Results suggested that TPH1 plays an important role in regulating calcium homeostasis via PTHrP and calcium transportation related factors in GMEC.

5-羟色胺(5-hydroxytryptamine,5-HT)是一种生物胺和色氨酸的衍生物,可以在动物中枢神经系统和多种外周组织合成,是调节机体生命活动的重要分子.外周组织中合成的5-HT作为一种激素,可以通过自分泌和旁分泌途径影响机体的多种功能.5-HT的受体多样且具有组织和细胞差异性,外周5-HT通过作用于不同的受体参与机体稳态的调节.现综合近年来外周5-HT功能的相关研究,从外周5-HT在不同组织中的调控作用进行综述,旨在为改善母畜泌乳性能、维持骨密度、调节糖脂代谢及控制能量平衡等方面提供思路.

Serotonin (5-HT) is a monoamine and it could regulate cell growth by its receptors working on signaling pathways. 5-HTP is the precursor of 5-HT that help 5-HT synthesis. B cell leukemia/lymphoma 3 (Bcl-3) involved in cell death and proliferation through mitogen activated protein kinase (MAPK) pathway. However, there is little information about the effects of MAPK/Bcl-3 on apoptosis of goat mammary gland epithelial cells (GMECs). The aim of this study is to explore the interaction among 5-HTP, MAPK and Bcl-3 in GMEC apoptosis. In this study, 5-HTP treatment decreased cell apoptosis and promoted phosphorylation of ERK1/2 in GMEC. We also found that the activation and inhibition of ERK1/2 could affect GMEC apoptosis. The Annexin V-FITC/PI staining and western blotting results suggested that 5-HTP decreased GMEC apoptosis through ERK1/2 signaling pathway. And the results of RT-qPCR and western blotting demonstrated that both 5-HTP and ERK1/2 positively regulated Bcl-3 expression. Sum up all the results, we could draw the conclusion that 5-HTP decreased GMEC apoptosis through MAPK/ERK/Bcl-3 pathway.

5-Hydroxy-l-tryptophan (5-HTP) is the primary product that converts l-tryptophan into 5-hydroxytryptamine by a rate-limiting enzyme. Our previous study found that 5-HTP could promote the intracellular calcium level in goat mammary epithelial cells (GMECs). Herein, first, dairy goats were injected with 5-HTP or saline daily from 7 days before delivery, and the calcium level in colostrum of 5-HTP-injected goats was significantly higher than that of saline-injected goats. Moreover, miR-99a-3p expression was significantly increased after 5-HTP treatment from transcriptome sequencing analysis and quantitative real-time polymerase chain reaction. In addition, it was found that ATP2B1 is one of the target genes of miR-99a-3p predicted by bioinformatic methods, which plays a crucial role in the maintenance of intracellular calcium homeostasis of mammary epithelial cells. Next, we confirmed that miR-99a-3p could increase the intracellular calcium level via decreasing ATP2B1 in GMECs. Taken together, we draw the conclusion that 5-HTP promotes the calcium level in colostrum possibly by increasing intracellular calcium of mammary epithelial cells induced by the miR-99a-3p/ATP2B1 axis.

哺乳动物体内的钙代谢通常包括钙平衡和钙稳态.钙平衡是指体内总钙含量保持相对恒定的状态,钙稳态则指细胞内外的钙离子浓度保持稳定.肾脏、肠道、骨骼及雌性动物乳腺是动物体钙代谢的主要器官,其中存在着许多精细而复杂的调控网络.妊娠期钙代谢紊乱会严重影响母畜及胎儿的营养健康,维持体内钙代谢稳态对围产期哺乳动物尤为重要.本文综合近年来国内外妊娠期哺乳动物钙代谢相关研究,从不同组织器官钙代谢情况及钙代谢过程中重要的调节因子的调控作用进行综述,旨在为妊娠期哺乳动物钙代谢调控相关研究提供思路.

五羟色胺(5-hydroxytryphtamine,5-HT)是机体重要的单胺类神经递质,广泛分布于动物体各组织器官并参与机体多种生理功能.色氨酸羟化酶(tryptophan hydroxylase,TPH)1是5-HT合成的限速酶,在5-HT调控机体代谢过程中起关键作用.本研究旨在利用CRISPR/CAS9技术建立稳定敲除TPH1基因的山羊(Capra hircus)乳腺上皮细胞系,为研究山羊乳腺中5-HT调控山羊乳腺上皮细胞(goat mammaryepithelial cells,GMEC)钙离子代谢的分子机理提供实验材料.首先根据GenBank中山羊TPH1基因序列(GenBank No.:102184739),设计靶向TPH1基因第一外显子的单导链RNA (single guide RNA,sgRNA),用pX458和pX459分别构建重组真核表达载体pX458-sgRNA和pX459-sgRNA,将重组质粒转染山羊乳腺上皮细胞,使用嘌呤霉素(1 tg/mL)进行阳性细胞筛选,挑取单克隆细胞进行培养,用Western blot、T7E1酶切、基因组DNA测序等方法鉴定基因敲除效果.结果显示:通过CRISPR/CAS9技术成功在TPH1基因第一外显子打靶产生10bp碱基缺失.本研究成功构建并筛选获得了TPH1基因稳定敲除的单克隆山羊乳腺上皮细胞系,为5-HT调控乳腺生理的功能研究提供了重要材料.

Stearoyl-CoA desaturase 1 (SCD1) is a fatty acid desaturase catalyzing cis-double-bond formation in the Delta 9 position to produce monounsaturated fatty acids essential for the synthesis of milk fat. Previous studies using RNAi methods have provided support for a role of SCD1 in goat mammary epithelial cells (GMEC); however, RNAi presents several limitations that might preclude a truthful understanding of the biological function of SCD1. To explore the function of SCD1 on fatty acid metabolism in GMEC, we used CRISPR-Cas9-mediated SCD1 knockout through non-homologous end-joining (NHEJ) and homology-directed repair (HDR) pathways in GMEC. We successfully introduced nucleotide deletions and mutations in the SCD1 gene locus through the NHEJ pathway and disrupted its second exon via insertion of an EGFP-PuroR segment using the HDR pathway. In clones derived from the latter, gene- and protein-expression data indicated that we obtained a monoallelic SCD1 knockout. A T7EN1-mediated assay revealed no off-targets in the surveyed sites. The contents of triacylglycerol and cholesterol and the desaturase index were significantly decreased as a consequence of SCD1 knockout. The deletion of SCD1 decreased the expression of other genes involved in de novo fatty acid synthesis, including SREBFI and FASN, as well the fatty acid transporters FABP3 and FABP4. The downregulation of these genes partly explains the decrease of intracellular triacylglycerols. Our results indicate a successful SCD1 knockout in goat mammary cells using CRISPR-Cas9. The demonstration of the successful use of CRISPR-Cas9 in GMEC is an important step to producing transgenic goats to study mammary biology in vivo.